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Sample records for myosin heavy chains

  1. Heavy chain of Acanthamoeba myosine IB is a fusion of myosin-like and non-myosin-like sequences

    SciTech Connect

    Jung, G.; Korn, E.D.; Hammer, J.A. III

    1987-10-01

    Acanthamoeba castellanii myosins IA and IB demonstrate the catalytic properties of a myosin and can support analogues of contractile and motile activity in vitro, but their single, low molecular weight heavy chains, roughly globular shapes, and inabilities to self-assemble into filaments make them structurally atypical myosins. The authors present the complete amino acid sequence of the 128-kDa myosin IB heavy chain, which they deduced from the nucleotide sequence of the gene and which reveals that the polypeptide is a fusion of myosin-like and non-myosin-like sequences. Specifically, the amino-terminal approx. 76 kDa of amino acid sequence is highly similar to the globular head sequences of conventional myosins. By contrast, the remaining approx. 51 kDa of sequence shows no similarity to any portion of conventional myosin sequences, contains regions that are rich in glycine, proline, and alanine residues, and lacks the distinctive sequence characteristics of an ..cap alpha..-helical, coiled-coil structure. They conclude, therefore, that the protein is composed of a myosin globular head fused not to the typical coiled-coil rod-like myosin tail structure but rather to an unusual carboxyl-terminal domain. These results support the conclusion that filamentous myosin is not required for force generation and provide a further perspective on the structural requirements for myosin function. Finally, they find a striking conservation of intron/exon structure between this gene and a vertebrate muscle myosin gene. They discuss this observation in relation to the evolutionary origin of the myosin IB gene and the antiquity of myosin gene intron/exon structure.

  2. Arginylation of myosin heavy chain regulates skeletal muscle strength

    PubMed Central

    Cornachione, Anabelle S.; Leite, Felipe S.; Wang, Junling; Leu, Nicolae A.; Kalganov, Albert; Volgin, Denys; Han, Xuemei; Xu, Tao; Cheng, Yu-Shu; Yates, John R. R.; Rassier, Dilson E.; Kashina, Anna

    2014-01-01

    Protein arginylation is a post-translational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 knockout driven by skeletal muscle-specific creatine kinase (Ckmm) promoter. Such Ckmm-Ate1 mice were viable and outwardly normal, however their skeletal muscle strength was significantly reduced compared to the control. Mass spectrometry of the isolated skeletal myofibrils showed a limited set of proteins arginylated on specific sites, including myosin heavy chain. Atomic force microscopy measurements of the contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of the myosin filaments could be fully rescued by re-arginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in the muscle and exerts a direct effect on muscle strength through arginylation of myosin. PMID:25017061

  3. Masticatory (;superfast') myosin heavy chain and embryonic/atrial myosin light chain 1 in rodent jaw-closing muscles.

    PubMed

    Reiser, Peter J; Bicer, Sabahattin; Chen, Qun; Zhu, Ling; Quan, Ning

    2009-08-01

    Masticatory myosin is widely expressed among several vertebrate classes. Generally, the expression of masticatory myosin has been associated with high bite force for a carnivorous feeding style (including capturing/restraining live prey), breaking down tough plant material and defensive biting in different species. Masticatory myosin expression in the largest mammalian order, Rodentia, has not been reported. Several members of Rodentia consume large numbers of tree nuts that are encased in very hard shells, presumably requiring large forces to access the nutmeat. We, therefore, tested whether some rodent species express masticatory myosin in jaw-closing muscles. Myosin isoform expression in six Sciuridae species was examined, using protein gel electrophoresis, immunoblotting, mass spectrometry and RNA analysis. The results indicate that masticatory myosin is expressed in some Sciuridae species but not in other closely related species with similar diets but having different nut-opening strategies. We also discovered that the myosin light chain 1 isoform associated with masticatory myosin heavy chain, in the same four Sciuridae species, is the embryonic/atrial isoform. We conclude that rodent speciation did not completely eliminate masticatory myosin and that its persistent expression in some rodent species might be related to not only diet but also to feeding style. PMID:19648394

  4. Continued Expression of Neonatal Myosin Heavy Chain in Adult Dystrophic Skeletal Muscle

    NASA Astrophysics Data System (ADS)

    Bandman, Everett

    1985-02-01

    The expression of myosin heavy chain isoforms was examined in normal and dystrophic chicken muscle with a monoclonal antibody specific for neonatal myosin. Adult dystrophic muscle continued to contain neonatal myosin long after it disappeared from adult normal muscle. A new technique involving western blotting and peptide mapping demonstrated that the immunoreactive myosin in adult dystrophic muscle was identical to that found in neonatal normal muscle. Immunocytochemistry revealed that all fibers in the dystrophic muscle failed to repress neonatal myosin heavy chain. These studies suggest that muscular dystrophy inhibits the myosin gene switching that normally occurs during muscle maturation.

  5. Adaptations in myosin heavy chain profile in chronically unloaded muscles

    NASA Technical Reports Server (NTRS)

    Talmadge, R. J.; Roy, R. R.; Bodine-Fowler, S. C.; Pierotti, D. J.; Edgerton, V. R.

    1995-01-01

    In this review, myosin heavy chain (MHC) adaptations in response to several models of decreased neuromuscular activity (i.e. electrical activation and loading of a muscle) are evaluated. In each of these "reduced-activity" models it is important to: a) quantify the changes in electrical activation of the muscle as a result of the intervention; b) quantify the forces generated by the muscle; and c) determine whether the neuromuscular junction remains normal. Most of the models, including spaceflight, hindlimb suspension, spinal cord isolation, spinal cord transection, denervation, and limb immobilization in a shortened position, result in increases in the percentage of fast MHCs (or fast MHC mRNA) in normally slow rat muscles. It also can be inferred from histochemical data that increases in fast MHCs occur with TTX application and bed rest. The only "reduced-activity" model to consistently increase slow muscle myosin mRNA, and slow fibers is limb immobilization in a stretched position; however, this model results in at least a temporary increase in tension. It appears that the most common feature of these models that might induce MHC adaptations is the modification in loading rather than a change in the neuromuscular activity.

  6. Improving human skeletal muscle myosin heavy chain fiber typing efficiency.

    PubMed

    Murach, Kevin A; Bagley, James R; McLeland, Kathryn A; Arevalo, Jose A; Ciccone, Anthony B; Malyszek, Kylie K; Wen, Yuan; Galpin, Andrew J

    2016-04-01

    Single muscle fiber sodium dodecyl sulfate polyacrylamide gel-electrophoresis (SDS-PAGE) is a sensitive technique for determining skeletal muscle myosin heavy chain (MHC) composition of human biopsy samples. However, the number of fibers suitable to represent fiber type distribution via this method is undefined. Muscle biopsies were obtained from the vastus lateralis (VL) of nine resistance-trained males (25 ± 1 year, height = 179 ± 5 cm, mass = 82 ± 8 kg). Single fiber MHC composition was determined via SDS-PAGE. VL fiber type distribution [percent MHC I, I/IIa, IIa, IIa/IIx, and total "hybrids" (i.e. I/IIa + IIa/IIx)] was evaluated according to number of fibers analyzed per person (25 vs. 125). VL fiber type distribution did not differ according to number of fibers analyzed (P > 0.05). VL biopsy fiber type distribution of nine subjects is represented by analyzing 25 fibers per person. These data may help minimize cost, personnel-time, and materials associated with this technique, thereby improving fiber typing efficiency in humans. PMID:26842420

  7. Myosin heavy chain pattern in the Akhal-Teke horses.

    PubMed

    Leisson, K; Alev, K; Kaasik, P; Jaakma, Ü; Seene, T

    2011-04-01

    This study investigates the myosin heavy chain (MyHC) isoform composition in the gluteus medius muscle of the Akhal-Teke horses using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Fifteen horses aged between 1.5 and 23.5 years were used in this study and divided into three age groups: 1.5 to 4 (n = 6), 9 to 13 (n = 5) and 18.5 to 23.5 years (n = 4). The average content of the MyHC I isoform was 11.72 ± 1.07% (variation between individuals: 7.09% to 20.14%). The relative content of the MyHC IIa and IIx isoforms was subsequently 38.20 ± 1.46% (30.73% to 48.78%) and 50.07 ± 1.10% (43.8% to 56.78%) from the total MyHC. The MyHC pattern in the skeletal muscles of the Akhal-Teke horses shows that the muscles of these horses have a high capacity both for endurance and speed. PMID:22439988

  8. CARBONYLATION OF MYOSIN HEAVY CHAINS IN RAT HEARTS DURING DIABETES

    PubMed Central

    Shao, Chun-Hong; Rozanski, George J.; Nagai, Ryoji; Stockdale, Frank E.; Patel, Kaushik P.; Wang, Mu; Singh, Jaipaul; Mayhan, William G.; Bidasee, Keshore R.

    2010-01-01

    Cardiac inotropy progressively declines during diabetes mellitus. To date, the molecular mechanisms underlying this defect remain incompletely characterized. This study tests the hypothesis that ventricular myosin heavy chains (MHC) undergo carbonylation by reactive carbonyl species (RCS) during diabetes and these modifications contribute to the inotropic decline. Male Sprague-Dawley rats were injected with streptozotocin (STZ). Fourteen days later animals were divided into two groups: one group was treated with the RCS blocker aminoguanidine for six weeks, while the other group received no treatment. After eight weeks of diabetes, cardiac ejection fraction, fractional shortening, left ventricular pressure development (+dP/dt) and myocyte shortening were decreased by 9%, 16%, 34% and 18%, respectively. Ca2+- and Mg2+-actomyosin ATPase activities and peak actomyosin syneresis were also reduced by 35%, 28%, and 72%. MHC-α to MHC-β ratio was 12:88. Mass spectrometry and Western blots revealed the presence of carbonyl adducts on MHC-α and MHC-β. Aminoguandine treatment did not alter MHC composition, but it blunted formation of carbonyl adducts and decreases in actomyosin Ca2+-sensitive ATPase activity, syneresis, myocyte shortening, cardiac ejection fraction, fractional shortening and +dP/dt induced by diabetes. From these new data it can be concluded that in addition to isozyme switching, modification of MHC by RCS also contributes to the inotropic decline seen during diabetes. PMID:20359464

  9. Characterization of human cardiac myosin heavy chain genes

    SciTech Connect

    Yamauchi-Takihara, K.; Sole, M.J.; Liew, J.; Ing, D.; Liew, C.C. )

    1989-05-01

    The authors have isolated and analyzed the structure of the genes coding for the {alpha} and {beta} forms of the human cardiac myosin heavy chain (MYHC). Detailed analysis of four overlapping MYHC genomic clones shows that the {alpha}-MYHC and {beta}-MYHC genes constitute a total length of 51 kilobases and are tandemly linked. The {beta}-MYHC-encoding gene, predominantly expressed in the normal human ventricle and also in slow-twitch skeletal muscle, is located 4.5 kilobases upstream of the {alpha}-MYHC-encoding gene, which is predominantly expressed in normal human atrium. The authors have determined the nucleotide sequences of the {beta} form of the MYHC gene, which is 100% homologous to the cardiac MYHC cDNA clone (pHMC3). It is unlikely that the divergence of a few nucleotide sequences from the cardiac {beta}-MYHC cDNA clone (pHMC3) reported in a MYHC cDNA clone (PSMHCZ) from skeletal muscle is due to a splicing mechanism. This finding suggests that the same {beta} form of the cardiac MYHC gene is expressed in both ventricular and slow-twitch skeletal muscle. The promoter regions of both {alpha}- and {beta}-MYHC genes, as well as the first four coding regions in the respective genes, have also been sequenced. The sequences in the 5{prime}-flanking region of the {alpha}- and {beta}-MYHC-encoding genes diverge extensively from one another, suggesting that expression of the {alpha}- and {beta}-MYHC genes is independently regulated.

  10. Differential regulation of myosin heavy chains defines new muscle domains in zebrafish

    PubMed Central

    Nord, Hanna; Burguiere, Anne-Cecile; Muck, Joscha; Nord, Christoffer; Ahlgren, Ulf; von Hofsten, Jonas

    2014-01-01

    Numerous muscle lineages are formed during myogenesis within both slow- and fast-specific cell groups. In this study, we show that six fast muscle–specific myosin heavy chain genes have unique expression patterns in the zebrafish embryo. The expression of tail-specific myosin heavy chain (fmyhc2.1) requires wnt signaling and is essential for fast muscle organization within the tail. Retinoic acid treatment results in reduced wnt signaling, which leads to loss of the fmyhc2.1 domain. Retinoic acid treatment also results in a shift of muscle identity within two trunk domains defined by expression of fmyhc1.2 and fmyhc1.3 in favor of the anteriormost myosin isoform, fmyhc1.2. In summary, we identify new muscle domains along the anteroposterior axis in the zebrafish that are defined by individual nonoverlapping, differentially regulated expression of myosin heavy chain isoforms. PMID:24523292

  11. Quantitative determination of type I myosin heavy chain in bovine muscle with anti myosin monoclonal antibodies.

    PubMed

    Picard, B; Leger, J; Robelin, J

    1994-01-01

    Bovine type I muscle fibers were characterized by enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody specific for slow myosin heavy chains (MHC 1). Two bovine muscles, the Masseter and Cutaneus trunci, were analyzed by different complementary techniques: electrophoresis, immunoblotting and immunohistiology. The results showed that the two muscles have extreme characteristics. The Masseter contains only slow MHC and the Cutaneus trunci is composed solely of rapid MHC (MHC 2a and 2b). A standard for this ELISA was obtained by mixing the two muscles and was used as a reference in the determination of the percentage of MHC 1 in a given muscle. In this study, the Longissimus thoracis of 27 Charolais cattle were examined. The different conditions under which assays were carried out were described and the accuracy of the measurement was calculated. In view of the results, ELISA was chosen for the analysis of muscle fiber types in large numbers of animal specimens. This technique could be used in several research projects to study the muscle characteristics that determine beef quality. PMID:22061628

  12. Ozz-E3 ubiquitin ligase targets sarcomeric embryonic myosin heavy chain during muscle development.

    PubMed

    Campos, Yvan; Qiu, Xiaohui; Zanoteli, Edmar; Moshiach, Simon; Vergani, Naja; Bongiovanni, Antonella; Harris, A John; d'Azzo, Alessandra

    2010-01-01

    Muscle contractile proteins are expressed as a series of developmental isoforms that are in constant dynamic remodeling during embryogenesis, but how obsolete molecules are recognized and removed is not known. Ozz is a developmentally regulated protein that functions as the adaptor component of a RING-type ubiquitin ligase complex specific to striated muscle. Ozz(-/-) mutants exhibit defects in myofibrillogenesis and myofiber differentiation. Here we show that Ozz targets the rod portion of embryonic myosin heavy chain and preferentially recognizes the sarcomeric rather than the soluble pool of myosin. We present evidence that Ozz binding to the embryonic myosin isoform within sarcomeric thick filaments marks it for ubiquitination and proteolytic degradation, allowing its replacement with neonatal or adult isoforms. This unique function positions Ozz within a system that facilitates sarcomeric myosin remodeling during muscle maturation and regeneration. Our findings identify Ozz-E3 as the ubiquitin ligase complex that interacts with and regulates myosin within its fully assembled cytoskeletal structure. PMID:20352047

  13. Solution NMR assignment of the heavy chain complex of the human cardiac myosin regulatory light chain.

    PubMed

    Rostkova, Elena; Gautel, Mathias; Pfuhl, Mark

    2015-04-01

    The regulatory light chain (RLC) of striated and cardiac muscle myosin plays a complex role in muscle function and regulation. Together with the essential light chain it provides stability to the lever arm, which is essential for force generation. Furthermore, phosphorylation and interaction with myosin binding protein C (MyBP-C) suggest an additional role in the regulation of muscle contraction. The former is of particular importance in the heart, where RLC phosphorylation appears to be correlated to the wringing motion of heart contraction. To address these questions and because of a lack of mammalian RLC structures, we initiated an NMR study of the human cardiac regulatory myosin light chain. PMID:24414277

  14. Myosin light-chain phosphatase.

    PubMed Central

    Morgan, M; Perry, S V; Ottaway, J

    1976-01-01

    1. A method for the isolation of a new enzyme, myosin light-chain phosphatase, from rabbit white skeletal muscle by using a Sepharose-phosphorylated myosin light-chain affinity column is described. 2. The enzyme migrated as a single component on electrophoresis in sodium dodecyl sulphate/polyacrylamide gel at pH7.0, with apparent mol.wt. 70000. 3. The enzyme was highly specific for the phosphorylated P-light chain of myosin, had pH optima at 6.5 and 8.0 and was not inhibited by NaF. 4. A Ca2+-sensitive 'ATPase' (adenosine triphosphatase) system consisting of myosin light-chain kinase, myosin light-chain phosphatase and the P-light chain is described. 5. Evidence is presented for a phosphoryl exchange between Pi, phosphorylated P-light chain and myosin light-chain phosphatase. 6. Heavy meromyosin prepared by chymotryptic digestion can be phosphorylated by myosin light-chain kinase. 7. The ATPase activities of myosin and heavy meromyosin, in the presence and absence of F-actin, were not significantly changed (+/- 10%) by phosphorylation of the P-light chain. Images PLATE 1 PMID:186030

  15. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins

    SciTech Connect

    Maruta, H.; Korn, E.D.

    1981-01-10

    The heavy chains of Acanthamoeba myosins, IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with uv light at 0/sup 0/C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain.

  16. Myosin light chains: Teaching old dogs new tricks

    PubMed Central

    Heissler, Sarah M; Sellers, James R

    2014-01-01

    The myosin holoenzyme is a multimeric protein complex consisting of heavy chains and light chains. Myosin light chains are calmodulin family members which are crucially involved in the mechanoenzymatic function of the myosin holoenzyme. This review examines the diversity of light chains within the myosin superfamily, discusses interactions between the light chain and the myosin heavy chain as well as regulatory and structural functions of the light chain as a subunit of the myosin holoenzyme. It covers aspects of the myosin light chain in the localization of the myosin holoenzyme, protein-protein interactions and light chain binding to non-myosin binding partners. Finally, this review challenges the dogma that myosin regulatory and essential light chain exclusively associate with conventional myosin heavy chains while unconventional myosin heavy chains usually associate with calmodulin. PMID:26155737

  17. Myosin heavy chain expression in rabbit masseter muscle during postnatal development.

    PubMed Central

    Bredman, J J; Weijs, W A; Korfage, H A; Brugman, P; Moorman, A F

    1992-01-01

    The expression of isoforms of myosin heavy chain (MHC) during postnatal development was studied in the masseter muscle of the rabbit. Evidence is presented that in addition to adult fast and slow myosin, the rabbit masseter contains neonatal and 'cardiac' alpha-MHC. During postnatal growth myosin transitions take place from neonatal and fast (IIA, IIA/IIB--referring to a fibre containing both IIA and IIB MHCs) MHC to adult 'cardiac' alpha-MHC and I/alpha-MHC. Since there is a temporary population of fibres containing IIA/alpha-MHC during the first 4 wk of development with a peak in the 3rd to 4th wk, the transition from IIA-MHC to alpha-MHC may occur in these IIA/alpha-MHC-containing fibres. The appearance of 'cardiac' alpha-MHC coincides with the timing of weaning, suggesting that the changes in MHC content, that probably result in a transition to a lower speed of contraction, have functional significance related to weaning. The finding of neonatal MHC in adult rabbits indicates that the masseter develops at a rate and in a way that is distinct from most other skeletal muscles. A spatiotemporal variation in expression of myosin isozymes within the masseter was observed, with many fibres containing more than one myosin type, indicating developmentally regulated spatial differences in function. Images Fig. 2 Fig. 3 Fig. 4 Fig. 7 PMID:1387129

  18. Myosin heavy chain-based fibre types in red cell hyper- and normovolaemic Standardbred trotters.

    PubMed

    Karlström, K; Essén-Gustavsson, B

    2002-09-01

    An assumed link between red cell hypervolaemia, an excessive amount of training and impaired performance of hypervolaemic horses has led to a theory that the muscle fibres could be affected. Myosin heavy chain (MHC)-based fibre type composition in gluteus medius muscle of red blood cell normo- (NV) and hypervolaemic (HV) Standardbred trotters was evaluated using immunohistochemistry. Muscle biopsies were obtained from 13 NV and 16 HV horses. Serial transverse sections were cut and reacted with antibodies against different isoforms of the myosin heavy chains MHCI, MHCIIA and MHCIIX. Sections were also stained for myofibrillar ATPase pH 4,6 to identify types I, IIA and IIB, and NADH tetrazolium reductase to evaluate the oxidative capacity. The results show that types I and IIA fibres corresponded between staining methods, whereas IIB fibres in the ATPase stains were more numerous than pure MHCIIX fibres from immunohistochemistry. Many fibres identified histochemically as type IIB fibres contained both MHC isoforms IIA and IIX (MHCIIAX). Most fibres had a high oxidative capacity, but among the fibres within a section, the lowest was seen subjectively in pure MHCIIX fibres. Immunohistochemical stains make it possible to detect differences in fibre type composition that are not observed with myosin ATPase stainings, as it was found that HV horses had a lower percentage of MHCIIX fibres than NV horses. Immunohistochemical methods are, therefore, valuable for use in further research and clinical studies concerning muscle adaptations. PMID:12405701

  19. Distribution of myosin heavy chain isoforms in muscular dystrophy: insights into disease pathology

    PubMed Central

    Beedle, Aaron M

    2016-01-01

    Myosin heavy chain isoforms are an important component defining fiber type specific properties in skeletal muscle, such as oxidative versus glycolytic metabolism, rate of contraction, and fatigability. While the molecular mechanisms that underlie specification of the different fiber types are becoming clearer, how this programming becomes disrupted in muscular dystrophy and the functional consequences of fiber type changes in disease are not fully resolved. Fiber type changes in disease, with specific focus on muscular dystrophies caused by defects in the dystrophin glycoprotein complex, are discussed. PMID:27430020

  20. Malalignment of the sarcomeric filaments in hypertrophic cardiomyopathy with cardiac myosin heavy chain gene mutation

    PubMed Central

    Muraishi, A; Kai, H; Adachi, K; Nishi, H; Imaizumi, T

    1999-01-01

    OBJECTIVE—To investigate changes in the alignment of the sarcomeric filaments in hypertrophic cardiomyopathy and the effects of cardiac β myosin heavy chain (β-MHC) mutation on the sarcomeric ultrastructure.
DESIGN—A retrospective analysis.
PATIENTS—Endomyocardial biopsy samples were examined by transmission electron microscopy in seven patients with hypertrophic cardiomyopathy and β-MHC mutation, six with hypertrophic cardiomyopathy but without the mutation, and five controls (with chest pain syndromes).
MAIN OUTCOME MEASURE—Alignment of the sarcomeric filaments and the distance between neighbouring thick myosin filaments.
RESULTS—In controls, cross sections of the sarcomere at the A band showed a highly organised orthohexagonal array with 6 thin actin filaments surrounding one thick myosin filament, whereas in hypertrophic cardiomyopathy the alignment of the sarcomeric filaments was sparse and disrupted. In hypertrophic cardiomyopathy with a mutation, the distance between neighbouring thick myosin filaments was greater than in controls (mean (SD) 45.3 (4.7) v 38.5 (3.5) nm, p < 0.05), and the variance of the distance was greater than in controls (8.0 (0.7) v 4.8 (1.0) nm, p < 0.001) or in patients with hypertrophic cardiomyopathy without a mutation (6.7 (0.6) nm, p < 0.05). In the latter, the variance of the distance was also greater than in the controls (p < 0.01). A significant correlation was found between the grade of the myocyte hypertrophy and the variance of the distance (r = 0.654; p < 0.01).
CONCLUSIONS—The alignment of the sarcomeric filaments is disrupted in hypertrophic cardiomyopathy, particularly when there is β-MHC mutation.


Keywords: hypertrophic cardiomyopathy; β myosin heavy chain; myosin filament; sarcomere PMID:10525522

  1. Myosin, Transgelin, and Myosin Light Chain Kinase

    PubMed Central

    Léguillette, Renaud; Laviolette, Michel; Bergeron, Celine; Zitouni, Nedjma; Kogut, Paul; Solway, Julian; Kachmar, Linda; Hamid, Qutayba; Lauzon, Anne-Marie

    2009-01-01

    Rationale: Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma. Objectives: We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax. Methods: We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay. Measurements and Main Results: We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion. Conclusions: Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma. PMID:19011151

  2. Myosin heavy chain composition in the rat diaphragm - Effect of age and exercise training

    NASA Technical Reports Server (NTRS)

    Gosselin, Luc E.; Betlach, Michael; Vailas, Arthur C.; Greaser, Marion L.; Thomas, D. P.

    1992-01-01

    The effects of aging and exercise training on the myosin heavy chain (MHC) composition were determined in both the costal and crural diaphragm regions of female Fischer 344 rats. Treadmill running at 75 percent maximal oxygen consumption resulted in similar increases in plantaris muscle citrate synthase activity in both young (5 mo) and old (23mo) trained animals (P less than 0.05). It was found that the ratio of fast to slow MHC was significantly higher (P less than 0.005) in the crural compared with costal diaphragm region in both age groups. A significant age-related increase in persentage of slow MHC was observed in both diaphragm regions. The relative proportion of slow MHC in either costal or crural region was not changed by exercise training.

  3. Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

    NASA Technical Reports Server (NTRS)

    Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

    1999-01-01

    Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

  4. Protective Effects of Clenbuterol against Dexamethasone-Induced Masseter Muscle Atrophy and Myosin Heavy Chain Transition

    PubMed Central

    Umeki, Daisuke; Ohnuki, Yoshiki; Mototani, Yasumasa; Shiozawa, Kouichi; Suita, Kenji; Fujita, Takayuki; Nakamura, Yoshiki; Saeki, Yasutake; Okumura, Satoshi

    2015-01-01

    Background Glucocorticoid has a direct catabolic effect on skeletal muscle, leading to muscle atrophy, but no effective pharmacotherapy is available. We reported that clenbuterol (CB) induced masseter muscle hypertrophy and slow-to-fast myosin heavy chain (MHC) isoform transition through direct muscle β2-adrenergic receptor stimulation. Thus, we hypothesized that CB would antagonize glucocorticoid (dexamethasone; DEX)-induced muscle atrophy and fast-to-slow MHC isoform transition. Methodology We examined the effect of CB on DEX-induced masseter muscle atrophy by measuring masseter muscle weight, fiber diameter, cross-sectional area, and myosin heavy chain (MHC) composition. To elucidate the mechanisms involved, we used immunoblotting to study the effects of CB on muscle hypertrophic signaling (insulin growth factor 1 (IGF1) expression, Akt/mammalian target of rapamycin (mTOR) pathway, and calcineurin pathway) and atrophic signaling (Akt/Forkhead box-O (FOXO) pathway and myostatin expression) in masseter muscle of rats treated with DEX and/or CB. Results and Conclusion Masseter muscle weight in the DEX-treated group was significantly lower than that in the Control group, as expected, but co-treatment with CB suppressed the DEX-induced masseter muscle atrophy, concomitantly with inhibition of fast-to-slow MHC isoforms transition. Activation of the Akt/mTOR pathway in masseter muscle of the DEX-treated group was significantly inhibited compared to that of the Control group, and CB suppressed this inhibition. DEX also suppressed expression of IGF1 (positive regulator of muscle growth), and CB attenuated this inhibition. Myostatin protein expression was unchanged. CB had no effect on activation of the Akt/FOXO pathway. These results indicate that CB antagonizes DEX-induced muscle atrophy and fast-to-slow MHC isoform transition via modulation of Akt/mTOR activity and IGF1 expression. CB might be a useful pharmacological agent for treatment of glucocorticoid

  5. Identification and genomic cloning of CMHC1. A unique myosin heavy chain expressed exclusively in the developing chicken heart.

    PubMed

    Croissant, J D; Carpenter, S; Bader, D

    2000-01-21

    We report the identification and cloning of a unique chick myosin heavy chain (CMHC1) that is expressed exclusively in the heart during embryogenesis. Using primers specific to myosin heavy chains, we used reverse transcriptase-polymerase chain reaction to clone and isolate CMHC1 from embryonic day 10 chicken heart RNA. Sequence analysis indicated that CMHC1 was a novel member of the myosin heavy chain family. Expression of the CMHC1 transcripts was detected in Hamburger Hamilton stage 10 chick embryos in the fusing myocardium. Expression of CMHC1 was maintained at high levels throughout the tubular heart of later stage embryos. Reverse transcriptase-polymerase chain reaction and in situ hybridizations failed to detect CMHC1 transcripts in the developing somites, limb buds, or skeletal musculature at any stage of chick development. Genomic CMHC1 clones have been isolated that contain sequences approximately 5.2 kilobase upstream of the presumptive CMHC1 transcription start site. Portions of the upstream regulatory region induced a 21-fold increase in reporter gene expression in primary cardiomyocytes. Because of its unique cardiac-restricted expression, CMHC1 will provide an excellent model system to study the molecular mechanisms required for the early developmental regulation of heart-specific genes. PMID:10636896

  6. Chronic sleep deprivation alters the myosin heavy chain isoforms in the masseter muscle in rats.

    PubMed

    Cao, Ruihua; Huang, Fei; Wang, Peihuan; Chen, Chen; Zhu, Guoxiong; Chen, Lei; Wu, Gaoyi

    2015-05-01

    To investigate the changes in myosin heavy chain (MyHC) isoforms of rat masseter muscle fibres caused by chronic sleep deprivation and a possible link with the pathogenesis of disorders of the temporomandibular joint (TMJ). A total of 180 male rats were randomly divided into three groups (n=60 in each): cage controls, large platform controls, and chronic sleep deprivation group. Each group was further divided into three subgroups with different observation periods (7, 14, and 21 days). We investigated he expression of MyHC isoforms in masseter muscle fibres by real-time quantitative polymerase chain reaction (PCR), Western blotting, and immunohistochemical staining. In rats with chronic sleep deprivation there was increased MyHC-I expression in layers of both shallow and deep muscles at 7 and 21 days compared with the control groups, whereas sleep deprivation was associated with significantly decreased MyHC-II expression. At 21 days, there were no differences in MyHC-I or MyHC-II expression between the groups and there were no differences between the two control groups at any time point. These findings suggest that chronic sleep deprivation alters the expression of MyHC isoforms, which may contribute to the pathogenesis of disorders of the TMJ. PMID:25804396

  7. Structural organization of the human cardiac [alpha]-myosin heavy chain gene (MYH6)

    SciTech Connect

    Epp, T.A.; Dixon, I.M.C.; Wang, H.Y.; Sole, M.J.; Liew, C.C. )

    1993-12-01

    The human myocardium expresses two cardiac myosin heavy chain (MyHC) isoforms, [alpha] and [beta], that exist in tandem array on chromosome 14q12. The authors have previously sequenced the entire human cardiac [beta]-MyHC gene and now report the complete nucleotide sequence of the human cardiac [alpha]-MyHC, encompassing 26,159 bp as well as the entire 4484-bp 5'-flanking intergenic region. The gene (MYH6) consists of 39 exons, 37 of which contain coding information. The 5'-untranslated region is split into 3 exons, with the third exon containing the AUG translocation initiation codon. With the exception of the 13th intron of the human cardiac [beta]-MyHC, which is not present within the [alpha]-isogene, all exon/intron boundaries are conserved. Conspicuous sequence motifs contained within the [alpha]-MyHC gene include four Alu repeats, a single (GT)[sub n] element, and a homopurine-homopyrimidine tract containing 23 GAA repeating units followed by 10 GAG repeating units. Comparison of the encoded amino acid sequence with a previously reported human [alpha]-MyHC cDNA sequence reveals several potential polymorphisms. 29 refs., 1 fig., 1 tab.

  8. Effects of inactivity on myosin heavy chain composition and size of rat soleus fibers

    NASA Technical Reports Server (NTRS)

    Grossman, E. J.; Roy, R. R.; Talmadge, R. J.; Zhong, H.; Edgerton, V. R.

    1998-01-01

    Myosin heavy chain (MHC) and fiber size properties of the adult rat soleus were determined after 4-60 days of complete inactivity, i.e., lumbar spinal cord isolation. Soleus atrophy was rapid and progressive, i.e., 25% and 64% decrease in weight and 33% and 75% decrease in fiber size after 4 and 60 days of inactivity, respectively. Changes in MHC occurred at a slower rate than the atrophic response. After 15 days there was de novo expression of type IIx MHC (approximately 10%). By 60 days, type IIx MHC accounted for 33% of the total MHC content, and 7% of the fibers contained only type IIx MHC. The relative amount of type I MHC was reduced from 93% in control to 49% after 60 days of inactivity. Therefore, the effects of 60 days of inactivity suggest that during this time period at least 75% of fiber size and approximately 40% of type I MHC composition of the adult rat soleus can be attributed to activation-related events.

  9. Hemiparetic Stroke Alters Vastus Lateralis Myosin Heavy Chain Profiles Between the Paretic and Nonparetic Muscles

    PubMed Central

    McKENZIE, MICHAEL J.; YU, SHUZHEN; PRIOR, STEVEN J.; MACKO, RICHARD F.; HAFER-MACKO, CHARLENE E.

    2010-01-01

    Skeletal muscle phenotype alterations following hemiparetic stroke contribute to disabilities associated with stroke. The phenotypic response following stroke is undefined. This investigation examined the myosin heavy chain (MHC) composition of the vastus lateralis (VL) of stroke survivors in paretic (P) and nonparetic (NP) muscle. Protein obtained from VL of 10 stroke survivors was isolated and purified, and MHC gel electrophoresis was performed. The MHC bands were quantified, and a paired sample two-tailed T test with significance set at p ≤ 0.05 was performed. MHC I expression was significantly less in P versus NP VL (.93 vs. 1.00 arbitrary units [AU]). Significantly more IIx MHC was found in the P versus NP VL (1.33 vs. 1.0). No significant differences in type IIa MHC (1.07 P vs. 1.00 NP) were found. These changes in MHC composition suggest an alteration in muscle function due to stroke or the altered activity patterns of muscle following stroke. PMID:19266390

  10. Single-fiber myosin heavy chain polymorphism during postnatal development: modulation by hypothyroidism

    NASA Technical Reports Server (NTRS)

    di Maso, N. A.; Caiozzo, V. J.; Baldwin, K. M.

    2000-01-01

    The primary objective of this study was to follow the developmental time course of myosin heavy chain (MHC) isoform transitions in single fibers of the rodent plantaris muscle. Hypothyroidism was used in conjunction with single-fiber analyses to better describe a possible linkage between the neonatal and fast type IIB MHC isoforms during development. In contrast to the general concept that developmental MHC isoform transitions give rise to muscle fibers that express only a single MHC isoform, the single-fiber analyses revealed a very high degree of MHC polymorphism throughout postnatal development. In the adult state, MHC polymorphism was so pervasive that the rodent plantaris muscles contained approximately 12-15 different pools of fibers (i.e., fiber types). The degree of polymorphism observed at the single-fiber level made it difficult to determine specific developmental schemes analogous to those observed previously for the rodent soleus muscle. However, hypothyroidism was useful in that it confirmed a possible link between the developmental regulation of the neonatal and fast type IIB MHC isoforms.

  11. Nonmuscle myosin heavy chain IIA mediates Epstein–Barr virus infection of nasopharyngeal epithelial cells

    PubMed Central

    Xiong, Dan; Du, Yong; Wang, Hong-Bo; Zhao, Bo; Zhang, Hua; Li, Yan; Hu, Li-Juan; Cao, Jing-Yan; Zhong, Qian; Liu, Wan-Li; Li, Man-Zhi; Zhu, Xiao-Feng; Tsao, Sai Wah; Hutt-Fletcher, Lindsey M.; Song, Erwei; Zeng, Yi-Xin; Kieff, Elliott; Zeng, Mu-Sheng

    2015-01-01

    EBV causes B lymphomas and undifferentiated nasopharyngeal carcinoma (NPC). Although the mechanisms by which EBV infects B lymphocytes have been extensively studied, investigation of the mechanisms by which EBV infects nasopharyngeal epithelial cells (NPECs) has only recently been enabled by the successful growth of B lymphoma Mo-MLV insertion region 1 homolog (BMI1)-immortalized NPECs in vitro and the discovery that neuropilin 1 expression positively affects EBV glycoprotein B (gB)-mediated infection and tyrosine kinase activations in enhancing EBV infection of BMI1-immortalized NPECs. We have now found that even though EBV infected NPECs grown as a monolayer at extremely low efficiency (<3%), close to 30% of NPECs grown as sphere-like cells (SLCs) were infected by EBV. We also identified nonmuscle myosin heavy chain IIA (NMHC-IIA) as another NPEC protein important for efficient EBV infection. EBV gH/gL specifically interacted with NMHC-IIA both in vitro and in vivo. NMHC-IIA densely aggregated on the surface of NPEC SLCs and colocalized with EBV. EBV infection of NPEC SLCs was significantly reduced by NMHC-IIA siRNA knock-down. NMHC-IIA antisera also efficiently blocked EBV infection. These data indicate that NMHC-IIA is an important factor for EBV NPEC infection. PMID:26290577

  12. Limited Expression of Slow Tonic Myosin Heavy Chain in Human Cranial Muscles

    PubMed Central

    Sokoloff, Alan J.; Li, Haiyan; Burkholder, Thomas J.

    2013-01-01

    Recent reports of slow tonic myosin heavy chain (MHCst) in human masticatory and laryngeal muscles suggest that MHCst may have a wider distribution in humans than previously thought. Because of the novelty of this finding, we sought to confirm the presence of MHCst in human masticatory and laryngeal muscles by reacting tissue from these muscles and controls from extraocular, intrafusal, cardiac, appendicular and developmental muscle with antibodies (Abs) ALD-58 and S46 considered highly specific for MHCst. At Ab dilutions producing minimal reaction to muscle fibers positive for MHCI, only extraocular, intrafusal and fetal tongue tissue reacted with Ab S46 had strong immunoreaction in an appreciable number of muscle fibers. In immunoblots Ab S46, but not Ab ALD-58, labeled adult extraocular muscles; no other muscles were labeled with either Ab. We conclude that, in humans, Ab S46 has greater specificity for MHCst than does Ab ALD-58. We suggest that reports of MHCst in human masticatory and laryngeal muscles reflect false-positive identification of MHCst due to cross-reactivity of Ab ALD-58 with another MHC isoform. PMID:17486578

  13. Effect of porcine Akirin2 on skeletal myosin heavy chain isoform expression.

    PubMed

    Chen, Xiaoling; Luo, Yanliu; Zhou, Bo; Huang, Zhiqing; Jia, Gang; Liu, Guangmang; Zhao, Hua; Yang, Zhouping; Zhang, Ruinan

    2015-01-01

    Akirin2 plays an important role in skeletal myogenesis. In this study, we found that porcine Akirin2 (pAkirin2) mRNA level was significantly higher in fast extensor digitorum longus (EDL) and longissimus lumborum (LL) muscles than in slow soleus (SOL) muscle of pigs. Overexpression of pAkirin2 increased the number of myosin heavy chain (MHC)-positive cells, indicating that pAkirin2 promoted myoblast differentiation. We also found that overexpression of pAkirin2 increased the mRNA expressions of MHCI and MHCIIa and decreased the mRNA expression of MHCIIb. Myocyte enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT) are the major downstream effectors of calcineurin. Here we also observed that the mRNA expressions of MEF2C and NFATc1 were notably elevated by pAkirin2 overexpression. Together, our data indicate that the role of pAkirin2 in modulating MHCI and MHCIIa expressions may be achieved through calcineurin/NFATc1 signaling pathway. PMID:25686036

  14. Effect of Porcine Akirin2 on Skeletal Myosin Heavy Chain Isoform Expression

    PubMed Central

    Chen, Xiaoling; Luo, Yanliu; Zhou, Bo; Huang, Zhiqing; Jia, Gang; Liu, Guangmang; Zhao, Hua; Yang, Zhouping; Zhang, Ruinan

    2015-01-01

    Akirin2 plays an important role in skeletal myogenesis. In this study, we found that porcine Akirin2 (pAkirin2) mRNA level was significantly higher in fast extensor digitorum longus (EDL) and longissimus lumborum (LL) muscles than in slow soleus (SOL) muscle of pigs. Overexpression of pAkirin2 increased the number of myosin heavy chain (MHC)-positive cells, indicating that pAkirin2 promoted myoblast differentiation. We also found that overexpression of pAkirin2 increased the mRNA expressions of MHCI and MHCIIa and decreased the mRNA expression of MHCIIb. Myocyte enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT) are the major downstream effectors of calcineurin. Here we also observed that the mRNA expressions of MEF2C and NFATc1 were notably elevated by pAkirin2 overexpression. Together, our data indicate that the role of pAkirin2 in modulating MHCI and MHCIIa expressions may be achieved through calcineurin/NFATc1 signaling pathway. PMID:25686036

  15. Control of myosin heavy chain expression: interaction of hypothyroidism and hindlimb suspension.

    PubMed

    Diffee, G M; Haddad, F; Herrick, R E; Baldwin, K M

    1991-12-01

    The aim of this study was to contrast competing influences, hypothyroidism and hindlimb suspension, on myosin heavy chain (MHC) expression studied at the protein level and mRNA level. Female Sprague-Dawley rats were assigned to either normal control (NC), normal suspended (NS), or hypothyroid (thyroidectomized) control (TC) and suspended (TS) groups. NS and TS animals were suspended for 14 days following which myofibrils and total RNA were purified from the hindlimb muscles. In the soleus and vastus intermedius (VI), there was an increase in type I MHC and a decrease in type IIa MHC in both the TC and TS groups and a decrease in type I and increase in type IIa MHC in the NS group. At the mRNA level, similar shifts were observed with the exception that 1) the increased type IIa MHC seen in the soleus and VI of the NS animals was not accompanied by an increase in IIa mRNA and 2) type IIb mRNA was increased in the NS soleus without concomitant changes in IIb protein levels. These data suggest the following: 1) a hypothyroid state predominates over mechanical unweighting factors in the control of MHC distribution in slow muscles; and 2) translational or posttranslational factors may be important in the regulation of type IIa and IIb MHC expression during hindlimb suspension. PMID:1767813

  16. Interaction of thyroid state and denervation on skeletal myosin heavy chain expression

    NASA Technical Reports Server (NTRS)

    Haddad, F.; Arnold, C.; Zeng, M.; Baldwin, K.

    1997-01-01

    The goal of this study was to examine the effects of altered thyroid state and denervation (Den) on skeletal myosin heavy chain (MHC) expression in the plantaris and soleus muscles. Rats were subjected to unilateral denervation (Den) and randomly assigned to one of three groups: (1) euthyroid; (2) hyperthyroid; (3) and hypothyroid. Denervation caused severe muscle atrophy and muscle-type specific MHC transformation. Denervation transformed the soleus to a faster muscle, and its effects required the presence of circulating thyroid hormone. In contrast, denervation transformed the plantaris to a slower muscle independently of thyroid state. Furthermore, thyroid hormone effects did not depend upon innervation status in the soleus, while they required the presence of the nerve in the plantaris. Collectively, these findings suggest that both thyroid hormone and intact nerve (a) differentially affect MHC transformations in fast and slow muscle; and (b) are important factors in regulating the optimal expression of both type I and IIB MHC genes. This research suggests that for patients with nerve damage and/or paralysis, both muscle mass and biochemical properties can also be affected by the thyroid state.

  17. Time course of myosin heavy chain transitions in neonatal rats: importance of innervation and thyroid state

    NASA Technical Reports Server (NTRS)

    Adams, G. R.; McCue, S. A.; Zeng, M.; Baldwin, K. M.

    1999-01-01

    During the postnatal period, rat limb muscles adapt to weight bearing via the replacement of embryonic (Emb) and neonatal (Neo) myosin heavy chains (MHCs) by the adult isoforms. Our aim was to characterize this transition in terms of the six MHC isoforms expressed in skeletal muscle and to determine the importance of innervation and thyroid hormone status on the attainment of the adult MHC phenotype. Neonatal rats were made hypothyroid via propylthiouracil (PTU) injection. In normal and PTU subgroups, leg muscles were unilaterally denervated at 15 days of age. The MHC profiles of plantaris (PLN) and soleus (Sol) muscles were determined at 7, 14, 23, and 30 days postpartum. At day 7, the Sol MHC profile was 55% type I, 30% Emb, and 10% Neo; in the PLN, the pattern was 60% Neo and 25% Emb. By day 30 the Sol and PLN had essentially attained an adult MHC profile in the controls. PTU augmented slow MHC expression in the Sol, whereas in the PLN it markedly repressed IIb MHC by retaining neonatal MHC expression. Denervation blunted the upregulation of IIb in the PLN and of Type I in the Sol and shifted the pattern to greater expression of IIa and IIx MHCs in both muscles. In contrast to previous observations, these findings collectively suggest that both an intact thyroid and innervation state are obligatory for the attainment of the adult MHC phenotype, particularly in fast-twitch muscles.

  18. Age dependence of myosin heavy chain transitions induced by creatine depletion in rat skeletal muscle

    NASA Technical Reports Server (NTRS)

    Adams, Gregory R.; Baldwin, Kenneth M.

    1995-01-01

    This study was designed to test the hypothesis that myosin heavy chain (MHC) plasticity resulting from creatine depletion is an age-dependent process. At weaning (age 28 days), rat pups were placed on either standard rat chow (normal diet juvenile group) or the same chow supplemented with 1% wt/wt of the creatine analogue beta-guanidinopropionic acid (creatine depletion juvenile (CDJ) group). Two groups of adult rats (age approximately 8 wk) were placed on the same diet regimens (normal diet adult and creatine depletion adult (CDA) groups). After 40 days (CDJ and normal diet juvenile groups) and 60 days (CDA and normal diet adult groups), animals were killed and several skeletal muscles were removed for analysis of creatine content or MHC ditribution. In the CDJ group, creatine depletion (78%) was accompanied by significant shifts toward expression of slower MHC isoforms in two slow and three fast skeletal muscles. In contrast, creatine depletion in adult animals did not result in similar shifts toward slow MHC isoform expression in either muscle type. The results of this study indicate that there is a differential effect of creatine depletion on MHC tranitions that appears to be age dependent. These results strongly suggest that investigators contemplating experimental designs involving the use of the creatine analogue beta-guanidinopropionic acid should consider the age of the animals to be used.

  19. Prognostic implications of novel beta cardiac myosin heavy chain gene mutations that cause familial hypertrophic cardiomyopathy.

    PubMed Central

    Anan, R; Greve, G; Thierfelder, L; Watkins, H; McKenna, W J; Solomon, S; Vecchio, C; Shono, H; Nakao, S; Tanaka, H

    1994-01-01

    Three novel beta cardiac myosin heavy chain (MHC) gene missense mutations, Phe513Cys, Gly716Arg, and Arg719Trp, which cause familial hypertrophic cardiomyopathy (FHC) are described. One mutation in exon 15 (Phe513Cys) does not alter the charge of the encoded amino acid, and affected family members have a near normal life expectancy. The Gly716Arg mutation (exon 19; charge change of +1) causes FHC in three family members, one of whom underwent transplantation for heart failure. The Arg719Trp mutation (exon 19; charge change of -1) was found in four unrelated FHC families with a high incidence of premature death and an average life expectancy in affected individuals of 38 yr. A comparable high frequency of disease-related deaths in four families with the Arg719Trp mutation suggests that this specific gene defect directly accounts for the observed malignant phenotype. Further, the significantly different life expectancies associated with the Arg719Trp vs. Phe513Cys mutation (P < 0.001) support the hypothesis that mutations which alter the charge of the encoded amino acid affect survival more significantly than those that produce a conservative amino acid change. Images PMID:8282798

  20. Myosin heavy chain is stabilized by BCL-2 interacting cell death suppressor (BIS) in skeletal muscle

    PubMed Central

    Hong, Jin; Park, Jun-Sub; Lee, Hyun; Jeong, Jaemin; Hyeon Yun, Hye; Yun Kim, Hye; Ko, Young-Gyu; Lee, Jeong-Hwa

    2016-01-01

    BCL-2 interacting cell death suppressor (BIS), which is ubiquitously expressed, has important roles in various cellular processes, such as apoptosis, the cellular stress response, migration and invasion and protein quality control. In particular, BIS is highly expressed in skeletal and cardiac muscles, and BIS gene mutations result in human myopathy. In this study, we show that mRNA and protein levels of BIS were markedly increased during skeletal myogenesis in C2C12 cells and mouse satellite cells. BIS knockdown did not prevent the early stage of skeletal myogenesis, but did induce muscle atrophy and a decrease in the diameter of myotubes. BIS knockdown significantly suppressed the expression level of myosin heavy chain (MyHC) without changing the expression levels of myogenic marker proteins, such as Mgn, Cav-3 and MG53. In addition, BIS endogenously interacted with MyHC, and BIS knockdown induced MyHC ubiquitination and degradation. From these data, we conclude that molecular association of MyHC and BIS is necessary for MyHC stabilization in skeletal muscle. PMID:27034027

  1. Myosin heavy chain composition of tiger (Panthera tigris) and cheetah (Acinonyx jubatus) hindlimb muscles.

    PubMed

    Hyatt, Jon-Philippe K; Roy, Roland R; Rugg, Stuart; Talmadge, Robert J

    2010-01-01

    Felids have a wide range of locomotor activity patterns and maximal running speeds, including the very fast cheetah (Acinonyx jubatas), the roaming tiger (Panthera tigris), and the relatively sedentary domestic cat (Felis catus). As previous studies have suggested a relationship between the amount and type of activity and the myosin heavy chain (MHC) isoform composition of a muscle, we assessed the MHC isoform composition of selected hindlimb muscles from these three felid species with differing activity regimens. Using gel electrophoresis, western blotting, histochemistry, and immunohistochemistry with MHC isoform-specific antibodies, we compared the MHC composition in the tibialis anterior, medial gastrocnemius (MG), plantaris (Plt), and soleus muscles of the tiger, cheetah, and domestic cat. The soleus muscle was absent in the cheetah. At least one slow (type I) and three fast (types IIa, IIx, and IIb) MHC isoforms were present in the muscles of each felid. The tiger had a high combined percentage of the characteristically slower isoforms (MHCs I and IIa) in the MG (62%) and the Plt (86%), whereas these percentages were relatively low in the MG (44%) and Plt (55%) of the cheetah. In general, the MHC isoform characteristics of the hindlimb muscles matched the daily activity patterns of these felids: the tiger has daily demands for covering long distances, whereas the cheetah has requirements for speed and power. PMID:19768738

  2. Analysis of myosin heavy chain mRNA expression by RT-PCR

    NASA Technical Reports Server (NTRS)

    Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

    1997-01-01

    An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

  3. Effect of hypothyroidism on myosin heavy chain expression in rat pharyngeal dilator muscles.

    PubMed

    Petrof, B J; Kelly, A M; Rubinstein, N A; Pack, A I

    1992-07-01

    Although the association between hypothyroidism and obstructive sleep apnea is well established, the effect of thyroid hormone deficiency on contractile proteins in pharyngeal dilator muscles responsible for maintaining upper airway patency is unknown. In the present study, the effects of hypothyroidism on myosin heavy chain (MHC) expression were examined in the sternohyoid, geniohyoid, and genioglossus muscles of adult rats (n = 20). The relative proportions of MHC isoforms present were determined using MHC-specific monoclonal antibodies and oligonucleotide probes. All control muscles showed a paucity of type I MHC fibers, with greater than 90% of fibers containing fast-twitch type II MHCs. In the genioglossus muscle, a population of non-IIa non-IIb fast-twitch type II fibers (putatively identified as type IIx MHC fibers) were detected. Hypothyroidism induced significant changes in MHC expression in all muscles studied. In the sternohyoid, type I fibers increased from 6.2 to 16.9%, whereas type IIa fibers increased from 25.9 to 30.7%. Type I fibers in the geniohyoid increased from 1.2 to 12.8%, whereas type IIa fibers increased from 34.1 to 42.7%. The genioglossus showed the smallest relative increase in type I expression but the greatest induction of type IIa MHC. None of the muscles examined demonstrated reinduction of embryonic or neonatal MHC in response to thyroid hormone deficiency. In summary, hypothyroidism alters the MHC profile of pharyngeal dilators in a muscle-specific manner. These changes may play a role in the pathogenesis of obstructive apnea in hypothyroid patients. PMID:1506366

  4. Muscle fiber type specific induction of slow myosin heavy chain 2 gene expression by electrical stimulation

    SciTech Connect

    Crew, Jennifer R.; Falzari, Kanakeshwari; DiMario, Joseph X.

    2010-04-01

    Vertebrate skeletal muscle fiber types are defined by a broad array of differentially expressed contractile and metabolic protein genes. The mechanisms that establish and maintain these different fiber types vary throughout development and with changing functional demand. Chicken skeletal muscle fibers can be generally categorized as fast and fast/slow based on expression of the slow myosin heavy chain 2 (MyHC2) gene in fast/slow muscle fibers. To investigate the cellular and molecular mechanisms that control fiber type formation in secondary or fetal muscle fibers, myoblasts from the fast pectoralis major (PM) and fast/slow medial adductor (MA) muscles were isolated, allowed to differentiate in vitro, and electrically stimulated. MA muscle fibers were induced to express the slow MyHC2 gene by electrical stimulation, whereas PM muscle fibers did not express the slow MyHC2 gene under identical stimulation conditions. However, PM muscle fibers did express the slow MyHC2 gene when electrical stimulation was combined with inhibition of inositol triphosphate receptor (IP3R) activity. Electrical stimulation was sufficient to increase nuclear localization of expressed nuclear-factor-of-activated-T-cells (NFAT), NFAT-mediated transcription, and slow MyHC2 promoter activity in MA muscle fibers. In contrast, both electrical stimulation and inhibitors of IP3R activity were required for these effects in PM muscle fibers. Electrical stimulation also increased levels of peroxisome-proliferator-activated receptor-{gamma} co-activator-1 (PGC-1{alpha}) protein in PM and MA muscle fibers. These results indicate that MA muscle fibers can be induced by electrical stimulation to express the slow MyHC2 gene and that fast PM muscle fibers are refractory to stimulation-induced slow MyHC2 gene expression due to fast PM muscle fiber specific cellular mechanisms involving IP3R activity.

  5. Sp3 proteins negatively regulate beta myosin heavy chain gene expression during skeletal muscle inactivity.

    PubMed

    Tsika, Gretchen; Ji, Juan; Tsika, Richard

    2004-12-01

    In adult skeletal muscle, beta myosin heavy chain (betaMyHC) gene expression is primarily restricted to slow type I fibers; however, its expression is down-regulated in response to muscle inactivity. Little is known about the signaling pathways and transcription factors that mediate this important functional response. This study demonstrates that increased binding of Sp3 to GC-rich elements in the betaMyHC promoter is a critical event in down-regulation of betaMyHC gene expression under non-weight-bearing conditions. Conversely, binding of Sp3 to these elements decreased while Sp1 binding increased with nuclear extracts from plantaris muscle exposed to mechanical overload, a stimulus that increases betaMyHC gene expression. In addition, these experiments revealed the existence of an Sp4-DNA binding complex when using adult skeletal muscle nuclear extract was used but not when nuclear extracts from cultured myotubes were used. Sp3 proteins are competitive inhibitors of Sp1-mediated betaMyHC reporter gene transactivation in both Drosophila SL-2 and mouse C2C12 myotubes. Sp4 is a weak activator of betaMyHC gene expression in SL-2 cells, which lack endogenous Sp1 activity, but does not activate betaMyHC gene expression in C2C12 myotubes, which have high levels of Sp1. These results suggest that competitive binding of Sp family proteins regulate betaMyHC gene transcription in response to altered neuromuscular activity. PMID:15572681

  6. Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity

    NASA Technical Reports Server (NTRS)

    Haddad, F.; Herrick, R. E.; Adams, G. R.; Baldwin, K. M.

    1993-01-01

    This study ascertained the effects of 9 days of zero gravity on the relative (percentage of total) and calculated absolute (mg/muscle) content of isomyosin expressed in both antigravity and locomotor skeletal muscle of ground control (CON) and flight-exposed (FL) rats. Results showed that although there were no differences in body weight between FL and CON animals, a significant reduction in muscle mass occurred in the vastus intermedius (VI) (P < 0.05) but not in the vastus lateralis (VL) or the tibialis anterior. Both total muscle protein and myofibril protein content were not different between the muscle regions examined in the FL and CON groups. In the VI, there were trends for reductions in the relative content of type I and IIa myosin heavy chains (MHCs) that were offset by increases in the relative content of both type IIb and possibly type IIx MHC protein (P > 0.05). mRNA levels were consistent with this pattern (P < 0.05). The same pattern held true for the red region of the VL as examined at both the protein and mRNA level (P < 0.05). When the atrophy process was examined, there were net reductions in the absolute content of both type I and IIa MHCs that were offset by calculated increases in type IIb MHC in both VI and red VL. Collectively, these findings suggest that there are both absolute and relative changes occurring in MHC expression in the "red" regions of antigravity skeletal muscle during exposure to zero gravity that could affect muscle function.

  7. Differential expression of caveolins and myosin heavy chains in response to forced exercise in rats

    PubMed Central

    Park, Sookyoung; Hong, Yunkyung; Lee, Youngjeon; Won, Jinyoung

    2012-01-01

    Exercise training can improve strength and lead to adaptations in the skeletal muscle and nervous systems. Skeletal muscles can develop into two types: fast and slow, depending on the expression pattern of myosin heavy chain (MHC) isoforms. Previous studies reported that exercise altered the distribution of muscle fiber types. It is not currently known what changes in the expression of caveolins and types of muscle fiber occur in response to the intensity of exercise. This study determined the changes in expression of caveolins and MHC type after forced exercise in muscular and non-muscular tissues in rats. A control (Con) group to which forced exercise was not applied and an exercise (Ex) group to which forced exercise was applied. Forced exercise, using a treadmill, was introduced at a speed of 25 m/min for 30 min, 3 times/day (07:00, 15:00, 23:00). Homogenized tissues were applied to extract of total RNA for further gene analysis. The expression of caveolin-3 and MHC2a in the gastrocnemius muscle of female rats significantly increased in the Ex group compared with the Con group (P<0.05). Furthermore, in the gastrocnemius muscle of male rats, the expression of MHC2x was significantly different between the two groups (P<0.05). There was an increased expression in caveolin-3 and a slightly decreased expression in TGFβ-1 in muscular tissues implicating caveolin-3 influences the expression of MHC isoforms and TGFβ-1 expression. Eventually, it implicates that caveolin-3 has positive regulatory function in muscle atrophy induced by neural dysfunction with spinal cord injury or stroke. PMID:22474468

  8. Myosin heavy-chain isoforms in the flight and leg muscles of hummingbirds and zebra finches.

    PubMed

    Velten, Brandy P; Welch, Kenneth C

    2014-06-01

    Myosin heavy chain (MHC) isoform complement is intimately related to a muscle's contractile properties, yet relatively little is known about avian MHC isoforms or how they may vary with fiber type and/or the contractile properties of a muscle. The rapid shortening of muscles necessary to power flight at the high wingbeat frequencies of ruby-throated hummingbirds and zebra finches (25-60 Hz), along with the varied morphology and use of the hummingbird hindlimb, provides a unique opportunity to understand how contractile and morphological properties of avian muscle may be reflected in MHC expression. Isoforms of the hummingbird and zebra finch flight and hindlimb muscles were electrophoretically separated and compared with those of other avian species representing different contractile properties and fiber types. The flight muscles of the study species operate at drastically different contraction rates and are composed of different histochemically defined fiber types, yet each exhibited the same, single MHC isoform corresponding to the chicken adult fast isoform. Thus, despite quantitative differences in the contractile demands of flight muscles across species, this isoform appears necessary for meeting the performance demands of avian powered flight. Variation in flight muscle contractile performance across species may be due to differences in the structural composition of this conserved isoform and/or variation within other mechanically linked proteins. The leg muscles were more varied in their MHC isoform composition across both muscles and species. The disparity in hindlimb MHC expression between hummingbirds and the other species highlights previously observed differences in fiber type composition and thrust production during take-off. PMID:24671242

  9. Force-velocity properties of human skeletal muscle fibres: myosin heavy chain isoform and temperature dependence.

    PubMed Central

    Bottinelli, R; Canepari, M; Pellegrino, M A; Reggiani, C

    1996-01-01

    1. A large population (n = 151) of human skinned skeletal muscle fibres has been studied. Force-velocity curves of sixty-seven fibres were obtained by load-clamp manoeuvres at 12 degrees C. In each fibre maximum shortening velocity (Vmax), maximum power output (Wmax), optimal velocity (velocity at which Wmax is developed, Vopt), optimal force (force at which Wmax is developed, Popt), specific tension (Po/CSA, isometric tension/cross-sectional area) were assessed. Unloaded shortening velocity (Vo) was also determined at 12 degrees C in a different group (n = 57) of fibres by slack-test procedure. 2. All fibres used for mechanical experiments were characterized on the basis of the myosin heavy chain (MHC) isoform composition by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and divided into five types: type I (or slow), types IIA and IIB (or fast), and types I-IIA and IIA-IIB (or mixed types). 3. Vmax, Wmax, Vopt, Popt, Vopt/Vmax ratio, Po/CSA and Vo were found to depend on MHC isoform composition. All parameters were significantly lower in type I than in the fast (type IIA and IIB) fibres. Among fast fibres, Vmax, Wmax, Vopt and Vo were significantly lower in type IIA and than in IIB fibres, whereas Popt, Po/CSA and Vopt/Vmax were similar. 4. The temperature dependence of Vo and Po/CSA was assessed in a group of twenty-one fibres in the range 12-22 degrees C. In a set of six fibres temperature dependence of Vmax was also studied. The Q10 (5.88) and activation energy E (125 kJ mol-1) values for maximum shortening velocity calculated from Arrhenius plots pointed to a very high temperature sensitivity. Po/CSA was very temperature dependent in the 12-17 degrees C range, but less dependent between 17 and 22 degrees C. Images Figure 1 Figure 3 Figure 6 PMID:8887767

  10. Proteomics Analysis of the Non-Muscle Myosin Heavy Chain IIa-Enriched Actin-Myosin Complex Reveals Multiple Functions within the Podocyte

    PubMed Central

    Hays, Thomas; Ma’ayan, Avi; Clark, Neil R.; Tan, Christopher M.; Teixeira, Avelino; Teixeira, Angela; Choi, Jae W.; Burdis, Nora; Jung, Sung Yun; Bajaj, Amol O.; O’Malley, Bert W.; He, John C.; Hyink, Deborah P.; Klotman, Paul E.

    2014-01-01

    MYH9 encodes non-muscle myosin heavy chain IIA (NMMHCIIA), the predominant force-generating ATPase in non-muscle cells. Several lines of evidence implicate a role for MYH9 in podocytopathies. However, NMMHCIIA‘s function in podocytes remains unknown. To better understand this function, we performed immuno-precipitation followed by mass-spectrometry proteomics to identify proteins interacting with the NMMHCIIA-enriched actin-myosin complexes. Computational analyses revealed that these proteins belong to functional networks including regulators of cytoskeletal organization, metabolism and networks regulated by the HIV-1 gene nef. We further characterized the subcellular localization of NMMHCIIA within podocytes in vivo, and found it to be present within the podocyte major foot processes. Finally, we tested the effect of loss of MYH9 expression in podocytes in vitro, and found that it was necessary for cytoskeletal organization. Our results provide the first survey of NMMHCIIA-enriched actin-myosin-interacting proteins within the podocyte, demonstrating the important role of NMMHCIIA in organizing the elaborate cytoskeleton structure of podocytes. Our characterization of NMMHCIIA’s functions goes beyond the podocyte, providing important insights into its general molecular role. PMID:24949636

  11. Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle

    NASA Technical Reports Server (NTRS)

    Baldwin, K. M.; Haddad, F.

    2001-01-01

    The goal of this mini-review is to summarize findings concerning the role that different models of muscular activity and inactivity play in altering gene expression of the myosin heavy chain (MHC) family of motor proteins in mammalian cardiac and skeletal muscle. This was done in the context of examining parallel findings concerning the role that thyroid hormone (T(3), 3,5,3'-triiodothyronine) plays in MHC expression. Findings show that both cardiac and skeletal muscles of experimental animals are initially undifferentiated at birth and then undergo a marked level of growth and differentiation in attaining the adult MHC phenotype in a T(3)/activity level-dependent fashion. Cardiac MHC expression in small mammals is highly sensitive to thyroid deficiency, diabetes, energy deprivation, and hypertension; each of these interventions induces upregulation of the beta-MHC isoform, which functions to economize circulatory function in the face of altered energy demand. In skeletal muscle, hyperthyroidism, as well as interventions that unload or reduce the weight-bearing activity of the muscle, causes slow to fast MHC conversions. Fast to slow conversions, however, are seen under hypothyroidism or when the muscles either become chronically overloaded or subjected to intermittent loading as occurs during resistance training and endurance exercise. The regulation of MHC gene expression by T(3) or mechanical stimuli appears to be strongly regulated by transcriptional events, based on recent findings on transgenic models and animals transfected with promoter-reporter constructs. However, the mechanisms by which T(3) and mechanical stimuli exert their control on transcriptional processes appear to be different. Additional findings show that individual skeletal muscle fibers have the genetic machinery to express simultaneously all of the adult MHCs, e.g., slow type I and fast IIa, IIx, and IIb, in unique combinations under certain experimental conditions. This degree of

  12. Analysis of Myosin Heavy Chain Isoforms from Longissimus Thoracis Muscle of Hanwoo Steer by Electrophoresis and LC-MS/MS

    PubMed Central

    2014-01-01

    The purpose of this study was to analyze myosin heavy chain (MHC) isoforms in bovine longissimus thoracis (LT) muscle by liquid chromatography (LC) and mass spectrometry (MS). LT muscles taken from Hanwoo (Korean native cattle) steer (n=3) used to separate myosin bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptide queries were obtained from the myosin bands by LC-MS/MS analysis following in-gel digestion with trypsin. A total of 33 and 43 queries were identified as common and unique peptides, respectively, of MHC isoforms (individual ions scores >43 indicate identity or extensive homology, p<0.05). MHC-1 (IIx), -2 (IIa), -4 (IIb), and -7 (slow/I) were identified based on the Mowse score (5118, 3951, 2526, and 2541 for MHC-1, -2, -4, and -7, respectively). However, more analysis is needed to confirm the expression of MHC-4 in bovine LT muscle because any query identified as a unique peptide of MHC-4 was not found. The queries that were identified as unique peptides could be used as peptide markers to confirm MHC-1 (14 queries), -2 (8 queries), and -7 (21 queries) in bovine LT muscle; no query identified as a unique peptide of MHC-4 was found. LC-MS/MS analysis is a useful approach to study MHC isoforms at the protein level. PMID:26761500

  13. Unexpectedly Low Mutation Rates in Beta-Myosin Heavy Chain and Cardiac Myosin Binding Protein Genes in Italian Patients With Hypertrophic Cardiomyopathy

    PubMed Central

    Roncarati, Roberta; Latronico, Michael VG; Musumeci, Beatrice; Aurino, Stefania; Torella, Annalaura; Bang, Marie-Louise; Jotti, Gloria Saccani; Puca, Annibale A; Volpe, Massimo; Nigro, Vincenzo; Autore, Camillo; Condorelli, Gianluigi

    2011-01-01

    Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. Fourteen sarcomeric and sarcomere-related genes have been implicated in HCM etiology, those encoding β-myosin heavy chain (MYH7) and cardiac myosin binding protein C (MYBPC3) reported as the most frequently mutated: in fact, these account for around 50% of all cases related to sarcomeric gene mutations, which are collectively responsible for approximately 70% of all HCM cases. Here, we used denaturing high-performance liquid chromatography followed by bidirectional sequencing to screen the coding regions of MYH7 and MYBPC3 in a cohort (n = 125) of Italian patients presenting with HCM. We found 6 MHY7 mutations in 9/125 patients and 18 MYBPC3 mutations in 19/125 patients. Of the three novel MYH7 mutations found, two were missense, and one was a silent mutation; of the eight novel MYBPC3 mutations, one was a substitution, three were stop codons, and four were missense mutations. Thus, our cohort of Italian HCM patients did not harbor the high frequency of mutations usually found in MYH7 and MYBPC3. This finding, coupled to the clinical diversity of our cohort, emphasizes the complexity of HCM and the need for more inclusive investigative approaches in order to fully understand the pathogenesis of this disease. J. Cell. Physiol. 226: 2894–2900, 2011. © 2011 Wiley-Liss, Inc. PMID:21302287

  14. Shared Gene Structures and Clusters of Mutually Exclusive Spliced Exons within the Metazoan Muscle Myosin Heavy Chain Genes

    PubMed Central

    Kollmar, Martin; Hatje, Klas

    2014-01-01

    Multicellular animals possess two to three different types of muscle tissues. Striated muscles have considerable ultrastructural similarity and contain a core set of proteins including the muscle myosin heavy chain (Mhc) protein. The ATPase activity of this myosin motor protein largely dictates muscle performance at the molecular level. Two different solutions to adjusting myosin properties to different muscle subtypes have been identified so far: Vertebrates and nematodes contain many independent differentially expressed Mhc genes while arthropods have single Mhc genes with clusters of mutually exclusive spliced exons (MXEs). The availability of hundreds of metazoan genomes now allowed us to study whether the ancient bilateria already contained MXEs, how MXE complexity subsequently evolved, and whether additional scenarios to control contractile properties in different muscles could be proposed, By reconstructing the Mhc genes from 116 metazoans we showed that all intron positions within the motor domain coding regions are conserved in all bilateria analysed. The last common ancestor of the bilateria already contained a cluster of MXEs coding for part of the loop-2 actin-binding sequence. Subsequently the protostomes and later the arthropods gained many further clusters while MXEs got completely lost independently in several branches (vertebrates and nematodes) and species (for example the annelid Helobdella robusta and the salmon louse Lepeophtheirus salmonis). Several bilateria have been found to encode multiple Mhc genes that might all or in part contain clusters of MXEs. Notable examples are a cluster of six tandemly arrayed Mhc genes, of which two contain MXEs, in the owl limpet Lottia gigantea and four Mhc genes with three encoding MXEs in the predatory mite Metaseiulus occidentalis. Our analysis showed that similar solutions to provide different myosin isoforms (multiple genes or clusters of MXEs or both) have independently been developed several times

  15. High fat/low carbohydrate diet attenuates left ventricular hypertrophy and prevents myosin heavy chain isoform switching induced by chronic hypertenstion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A switch in the expression of myosin heavy chain isoform (MHC) alpha to beta is observed with left ventricular hypertrophy (LVH) and heart failure. This switch is associated with a defect in myocardial energy production and contractile dysfunction. Similar MHC isoform profile is observed in the fe...

  16. Myosin Heavy Chain Gene Expression in Developing Neonatal Skeletal Muscle: Involvement of the Nerve, Gravity, and Thyroid State

    NASA Technical Reports Server (NTRS)

    Baldwin, K. M.; Adams, G.; Haddad, F.; Zeng, M.; Qin, A.; Qin, L.; McCue, S.; Bodell, P.

    1999-01-01

    The myosin heavy chain (MHC) gene family encodes at least six MHC proteins (herein designated as neonatal, embryonic, slow type I (beta), and fast IIa, IIx, and IIb) that are expressed in skeletal muscle in a muscle-specific and developmentally-regulated fashion. At birth, both antigravity (e.g. soleus) and locomotor (e.g., plantaris) skeletal muscles are undifferentiated relative to the adult MHC phenotype such that the neonatal and embryonic MHC isoforms account for 80 - 90% of the MHC pool in a fast locomotor muscle; whereas, the embryonic and slow, type I isoforms account for approx. 90% of the pool in a typical antigravity muscle. The goal of this study was to investigate the role of an intact nerve, gravity and thyroid hormone (T3), as well as certain interactions of these interventions, on MHC gene expression in developing neonatal skeletal muscles of rodents.

  17. Cardiac and skeletal myopathy in beta myosin heavy-chain simian virus 40 tsA58 transgenic mice.

    PubMed Central

    De Leon, J R; Federoff, H J; Dickson, D W; Vikstrom, K L; Fishman, G I

    1994-01-01

    The mechanisms regulating cardiac muscle differentiation and development are incompletely understood. To examine the relationships between cardiocyte proliferation and differentiation, we tested the ability of a fragment from the rat beta myosin heavy-chain (MHC beta) gene to correctly target expression of a thermolabile simian virus 40 large tumor antigen allele (tsA58) in the developing mouse. Transgene expression in the heart was observed as early as 10 days postconception and was developmentally regulated in parallel with the endogenous MHC beta gene. Expression was also detected in developing skeletal muscle, although at low levels. Despite the temperature sensitivity of the mutant large tumor antigen protein, a subset of transgenic mice in several lineages developed marked cardiac and skeletal myopathies. Images Fig. 2 Fig. 3 Fig. 4 PMID:8290557

  18. A Calcineurin-NFATc3-Dependent Pathway Regulates Skeletal Muscle Differentiation and Slow Myosin Heavy-Chain Expression

    PubMed Central

    Delling, Ulrike; Tureckova, Jolana; Lim, Hae W.; De Windt, Leon J.; Rotwein, Peter; Molkentin, Jeffery D.

    2000-01-01

    The differentiation and maturation of skeletal muscle cells into functional fibers is coordinated largely by inductive signals which act through discrete intracellular signal transduction pathways. Recently, the calcium-activated phosphatase calcineurin (PP2B) and the family of transcription factors known as NFAT have been implicated in the regulation of myocyte hypertrophy and fiber type specificity. Here we present an analysis of the intracellular mechanisms which underlie myocyte differentiation and fiber type specificity due to an insulinlike growth factor 1 (IGF-1)–calcineurin–NFAT signal transduction pathway. We demonstrate that calcineurin enzymatic activity is transiently increased during the initiation of myogenic differentiation in cultured C2C12 cells and that this increase is associated with NFATc3 nuclear translocation. Adenovirus-mediated gene transfer of an activated calcineurin protein (AdCnA) potentiates C2C12 and Sol8 myocyte differentiation, while adenovirus-mediated gene transfer of noncompetitive calcineurin-inhibitory peptides (cain or ΔAKAP79) attenuates differentiation. AdCnA infection was also sufficient to rescue myocyte differentiation in an IGF-depleted myoblast cell line. Using 10T1/2 cells, we demonstrate that MyoD-directed myogenesis is dramatically enhanced by either calcineurin or NFATc3 cotransfection, while a calcineurin inhibitory peptide (cain) blocks differentiation. Enhanced myogenic differentiation directed by calcineurin, but not NFATc3, preferentially specifies slow myosin heavy-chain expression, while enhanced differentiation through mitogen-activated protein kinase kinase 6 (MKK6) promotes fast myosin heavy-chain expression. These data indicate that a signaling pathway involving IGF-calcineurin-NFATc3 enhances myogenic differentiation whereas calcineurin acts through other factors to promote the slow fiber type program. PMID:10938134

  19. Noncompaction of the Ventricular Myocardium Is Associated with a De Novo Mutation in the β-Myosin Heavy Chain Gene

    PubMed Central

    Budde, Birgit S.; Binner, Priska; Waldmüller, Stephan; Höhne, Wolfgang; Blankenfeldt, Wulf; Hassfeld, Sabine; Brömsen, Jürgen; Dermintzoglou, Anastassia; Wieczorek, Marcus; May, Erik; Kirst, Elisabeth; Selignow, Carmen; Rackebrandt, Kirsten; Müller, Melanie; Goody, Roger S.; Vosberg, Hans-Peter; Nürnberg, Peter; Scheffold, Thomas

    2007-01-01

    Noncompaction of the ventricular myocardium (NVM) is the morphological hallmark of a rare familial or sporadic unclassified heart disease of heterogeneous origin. NVM results presumably from a congenital developmental error and has been traced back to single point mutations in various genes. The objective of this study was to determine the underlying genetic defect in a large German family suffering from NVM. Twenty four family members were clinically assessed using advanced imaging techniques. For molecular characterization, a genome-wide linkage analysis was undertaken and the disease locus was mapped to chromosome 14ptel-14q12. Subsequently, two genes of the disease interval, MYH6 and MYH7 (encoding the α- and β-myosin heavy chain, respectively) were sequenced, leading to the identification of a previously unknown de novo missense mutation, c.842G>C, in the gene MYH7. The mutation affects a highly conserved amino acid in the myosin subfragment-1 (R281T). In silico simulations suggest that the mutation R281T prevents the formation of a salt bridge between residues R281 and D325, thereby destabilizing the myosin head. The mutation was exclusively present in morphologically affected family members. A few members of the family displayed NVM in combination with other heart defects, such as dislocation of the tricuspid valve (Ebstein's anomaly, EA) and atrial septal defect (ASD). A high degree of clinical variability was observed, ranging from the absence of symptoms in childhood to cardiac death in the third decade of life. The data presented in this report provide first evidence that a mutation in a sarcomeric protein can cause noncompaction of the ventricular myocardium. PMID:18159245

  20. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    SciTech Connect

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev; Renkawitz-Pohl, Renate

    2013-02-15

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity.

  1. Dlc1 interaction with non-muscle myosin heavy chain II-A (Myh9) and Rac1 activation.

    PubMed

    Sabbir, Mohammad G; Dillon, Rachelle; Mowat, Michael R A

    2016-01-01

    The Deleted in liver cancer 1 (Dlc1) gene codes for a Rho GTPase-activating protein that also acts as a tumour suppressor gene. Several studies have consistently found that overexpression leads to excessive cell elongation, cytoskeleton changes and subsequent cell death. However, none of these studies have been able to satisfactorily explain the Dlc1-induced cell morphological phenotypes and the function of the different Dlc1 isoforms. Therefore, we have studied the interacting proteins associated with the three major Dlc1 transcriptional isoforms using a mass spectrometric approach in Dlc1 overexpressing cells. We have found and validated novel interacting partners in constitutive Dlc1-expressing cells. Our study has shown that Dlc1 interacts with non-muscle myosin heavy chain II-A (Myh9), plectin and spectrin proteins in different multiprotein complexes. Overexpression of Dlc1 led to increased phosphorylation of Myh9 protein and activation of Rac1 GTPase. These data support a role for Dlc1 in induced cell elongation morphology and provide some molecular targets for further analysis of this phenotype. PMID:26977077

  2. Isolation and characterization of an avian slow myosin heavy chain gene expressed during embryonic skeletal muscle fiber formation.

    PubMed

    Nikovits, W; Wang, G F; Feldman, J L; Miller, J B; Wade, R; Nelson, L; Stockdale, F E

    1996-07-19

    We have isolated and begun characterization of the quail slow myosin heavy chain (MyHC) 3 gene, the first reported avian slow MyHC gene. Expression of slow MyHC 3 in skeletal muscle is restricted to the embryonic period of development, when the fiber pattern of future fast and slow muscle is established. In embryonic hindlimb development, slow MyHC 3 gene expression coincides with slow muscle fiber formation as distinguished by slow MyHC-specific antibody staining. In addition to expression in embryonic appendicular muscle, slow MyHC 3 is expressed continuously in the atria. Transfection of slow MyHC 3 promoter-reporter constructs into embryonic myoblasts that form slow MyHC-expressing fibers identified two regions regulating expression of this gene in skeletal muscle. The proximal promoter, containing potential muscle-specific regulatory motifs, permits expression of a reporter gene in embryonic slow muscle fibers, while a distal element, located greater than 2600 base pairs upstream, further enhances expression 3-fold. The slow muscle fiber-restricted expression of slow MyHC 3 during embryonic development, and expression of slow MyHC 3 promoter-reporter constructs in embryonic muscle fibers in vitro, makes this gene a useful marker to study the mechanism establishing the slow fiber lineage in the embryo. PMID:8663323

  3. Fiber size, type, and myosin heavy chain content in rhesus hindlimb muscles after 2 weeks at 2 G

    NASA Technical Reports Server (NTRS)

    Tavakol, Morteza; Roy, Roland R.; Kim, Jung A.; Zhong, Hui; Hodgson, John A.; Hoban-Higgins, Tana M.; Fuller, Charles A.; Edgerton, V. Reggie

    2002-01-01

    BACKGROUND: Fiber atrophy and an increase in the percentage of fast fibers have been observed in Rhesus leg muscles after spaceflight. Hypothesis: Hypergravity will result in muscle fiber hypertrophy and an increase in the percentage of slow fibers. METHODS: Open muscle biopsies were obtained from Rhesus soleus, medial gastrocnemius (MG), and tibialis anterior (TA) muscles before and after 14 d of centrifugation (2 G) and in time-matched controls. Cage activity levels were measured by telemetry. RESULTS: Based on monoclonal antibody binding for myosin heavy chains (MHC), the fastest region of soleus contained a higher proportion of type I+II (27 vs. 13%) and had a tendency for a lower proportion of type I (38 vs. 61%, p = 0.10) fibers after than before centrifugation. There was a higher proportion of type I+II fibers in post- vs. pre-2 G (10 vs. 0.6%) MG biopsies. Fiber type distribution and MHC composition were unaffected in the TA. Overall, mean fiber sizes were unaffected by centrifugation. Average cage activity levels were 36% lower during than before 2 G. CONCLUSIONS: Our hypothesis was rejected. The changes in the proportion of fibers expressing type I MHC are the reverse of that expected with chronic loading of extensors and, paradoxically, are similar to changes observed with chronic unloading, such as occurs during spaceflight, in this primate model. The data are consistent with the observed decrease in total daily activity levels.

  4. Synergistic ablation does not affect atrophy or altered myosin heavy chain expression in the non-weight bearing soleus muscle

    NASA Technical Reports Server (NTRS)

    Linderman, J. K.; Talmadge, R. J.; Gosselink, K. L.; Tri, P. N.; Roy, R. R.; Grindeland, R. E.

    1996-01-01

    The purpose of this study was to investigate whether the soleus muscle undergoes atrophy and alterations in myosin heavy chain (MHC) composition during non-weight bearing in the absence of synergists. Thirty-two female rats were randomly assigned to four groups: control (C), synergistic ablation (ABL) of the gastrocnemius and plantaris muscles to overload the soleus muscle, hindlimb suspension (HLS), or a combination of synergistic ablation and hindlimb suspension (HLS-ABL). After 28 days of hindlimb suspension, soleus atrophy was more pronounced in HLS (58%) than in HLS-ABL (43%) rats. Compared to C rats, non-weight bearing decreased mixed and myofibrillar protein contents and Type I MHC 49%, 45%, and 7%, respectively, in HLS animals. In addition, de novo expression of fast Type IIx and Type IIb MHC (5% and 2%, respectively) was observed in HLS animals. Similarly, when compared to C rats, mixed and myofibrillar protein contents and Type I MHC decreased 43%, 46%, and 4%, respectively, in HLS-ABL animals. Also, de novo expression of Type IIx (4%) and IIb (1%) MHC was observed. Collectively, these data indicate that the loss of muscle protein and Type I MHC, and the de novo expression of Type IIx and Type IIb MHC in the rat soleus occur independently of the presence of synergists during non-weight bearing. Furthermore, these results confirm the contention that soleus mass and MHC expression are highly sensitive to alterations in mechanical load.

  5. Dlc1 interaction with non-muscle myosin heavy chain II-A (Myh9) and Rac1 activation

    PubMed Central

    Sabbir, Mohammad G.; Dillon, Rachelle; Mowat, Michael R. A.

    2016-01-01

    ABSTRACT The Deleted in liver cancer 1 (Dlc1) gene codes for a Rho GTPase-activating protein that also acts as a tumour suppressor gene. Several studies have consistently found that overexpression leads to excessive cell elongation, cytoskeleton changes and subsequent cell death. However, none of these studies have been able to satisfactorily explain the Dlc1-induced cell morphological phenotypes and the function of the different Dlc1 isoforms. Therefore, we have studied the interacting proteins associated with the three major Dlc1 transcriptional isoforms using a mass spectrometric approach in Dlc1 overexpressing cells. We have found and validated novel interacting partners in constitutive Dlc1-expressing cells. Our study has shown that Dlc1 interacts with non-muscle myosin heavy chain II-A (Myh9), plectin and spectrin proteins in different multiprotein complexes. Overexpression of Dlc1 led to increased phosphorylation of Myh9 protein and activation of Rac1 GTPase. These data support a role for Dlc1 in induced cell elongation morphology and provide some molecular targets for further analysis of this phenotype. PMID:26977077

  6. The influence of myosin heavy chain isoform content on mechanical behavior of the vastus lateralis in vivo.

    PubMed

    Trevino, Michael A; Herda, Trent J; Fry, Andrew C; Gallagher, Philip M; Vardiman, John P; Mosier, Eric M; Miller, Jonathan D

    2016-06-01

    This study examined correlations between type I percent myosin heavy chain isoform content (%MHC) and mechanomyographic amplitude (MMGRMS) during isometric muscle actions. Fifteen (age=21.63±2.39) participants performed 40% and 70% maximal voluntary contractions (MVC) of the leg extensors that included increasing, steady force, and decreasing segments. Muscle biopsies were collected and MMG was recorded from the vastus lateralis. Linear regressions were fit to the natural-log transformed MMGRMS-force relationships (increasing and decreasing segments) and MMGRMS was selected at the targeted force level during the steady force segment. Correlations were calculated among type I%MHC and the b (slopes) terms from the MMGRMS-force relationships and MMGRMS at the targeted force. For the 40% MVC, correlations were significant (P<0.02) between type I%MHC and the b terms from the increasing (r=-0.804) and decreasing (r=-0.568) segments, and MMGRMS from the steady force segment (r=-0.606). Type I%MHC was only correlated with MMGRMS during the steady force segment (P=0.044, r=-0.525) during the 70% MVC. Higher type I%MHC reduced acceleration in MMGRMS (b terms) during the 40% MVC and the amplitude during the steady force segments. The surface MMG signal recorded during a moderate intensity contraction provided insight on the contractile properties of the VL in vivo. PMID:27152756

  7. Mitochondrial and sarcoplasmic proteins, but not myosin heavy chain, are sensitive to leucine supplementation in old rat skeletal muscle.

    PubMed

    Guillet, Christelle; Zangarelli, Aude; Mishellany, Anne; Rousset, Paulette; Sornet, Claire; Dardevet, Dominique; Boirie, Yves

    2004-05-01

    Leucine has a major anabolic impact on muscle protein synthesis in young as in old animals. However, myosin heavy chain (MHC), sarcoplasmic and mitochondrial proteins may differently respond to anabolic factors, especially during aging. To test this hypothesis, fractional synthesis rates (FSR) of the three muscle protein fractions were measured using a flooding dose of [1-(13)C] phenylalanine, in gastrocnemius muscle of adult (8 months) and old (22 months) rats, either in postabsorptive state (PA), or 90-120 min after ingestion of a alanine-supplemented meal (PP+A) or a leucine-supplemented meal (PP+L). In adult and old rats, in comparison with PA, leucine stimulated mitochondrial (adult: 0.260+/-0.011 vs 0.238+/-0.012%h(-1); old: 0.289+/-0.010 vs 0.250+/-0.010%h(-1); PP+L vs PA, P<0.05) and sarcoplasmic (adult: 0.182+/-0.011 vs 0.143+/-0.006%h(-1); old: 0.195+/-0.010 vs 0.149+/-0.008%h(-1); PP+L vs PA, P<0.05) protein FSR, but not MHC synthesis in old rats (0.101+/-0.009 vs 0.137+/-0.018%h(-1); PP+L vs PA, P=NS). In conclusion, synthesis of specific muscle protein is activated by leucine supplementation, but MHC may be less sensitive to anabolic factors with aging. PMID:15130669

  8. Ablation of smooth muscle myosin heavy chain SM2 increases smooth muscle contraction and results in postnatal death in mice.

    PubMed

    Chi, Mei; Zhou, Yingbi; Vedamoorthyrao, Srikanth; Babu, Gopal J; Periasamy, Muthu

    2008-11-25

    The physiological relevance of smooth muscle myosin isoforms SM1 and SM2 has not been understood. In this study we generated a mouse model specifically deficient in SM2 myosin isoform but expressing SM1, using an exon-specific gene targeting strategy. The SM2 homozygous knockout (SM2(-/-)) mice died within 30 days after birth, showing pathologies including segmental distention of alimentary tract, retention of urine in renal pelvis, distension of bladder, and the development of end-stage hydronephrosis. In contrast, the heterozygous (SM2(+/-)) mice appeared normal and reproduced well. In SM2(-/-) bladder smooth muscle the loss of SM2 myosin was accompanied by a concomitant down-regulation of SM1 and a reduced number of thick filaments. However, muscle strips from SM2(-/-) bladder showed increased contraction to K(+) depolarization or in response to M3 receptor agonist Carbachol. An increase of contraction was also observed in SM2(-/-) aorta. However, the SM2(-/-) bladder was associated with unaltered regulatory myosin light chain (MLC20) phosphorylation. Moreover, other contractile proteins, such as alpha-actin and tropomyosin, were not altered in SM2(-/-) bladder. Therefore, the loss of SM2 myosin alone could have induced hypercontractility in smooth muscle, suggesting that distinctly from SM1, SM2 may negatively modulate force development during smooth muscle contraction. Also, because SM2(-/-) mice develop lethal multiorgan dysfunctions, we propose this regulatory property of SM2 is essential for normal contractile activity in postnatal smooth muscle physiology. PMID:19011095

  9. Abundant expression of myosin heavy-chain IIB RNA in a subset of human masseter muscle fibres

    PubMed Central

    Horton, Michael J.; Brandon, Carla A.; Morris, Terence J.; Braun, Thomas W.; Yaw, Kenneth M.; Sciote, James J.

    2013-01-01

    Type IIB fast fibres are typically demonstrated in human skeletal muscle by histochemical staining for the ATPase activity of myosin heavy-chain (MyHC) isoforms. However, the monoclonal antibody specific for the mammalian IIB isoform does not detect MyHC IIB protein in man and MyHC IIX RNA is found in histochemically identified IIB fibres, suggesting that the IIB protein isoform may not be present in man; if this is not so, jaw-closing muscles, which express a diversity of isoforms, are likely candidates for their presence. ATPase histochemistry, immunohistochemistry polyacrylamide gel electrophoresis and in situ hybridization, which included a MyHC IIB-specific mRNA riboprobe, were used to compare the composition and RNA expression of MyHC isoforms in a human jaw-closing muscle, the masseter, an upper limb muscle, the triceps, an abdominal muscle, the external oblique, and a lower limb muscle, the gastrocnemius. The external oblique contained a mixture of histochemically defined type I, IIA and IIB fibres distributed in a mosaic pattern, while the triceps and gastrocnemius contained only type I and IIA fibres. Typical of limb muscle fibres, the MyHC I-specific mRNA probes hybridized with histochemically defined type I fibres, the IIA-specific probes with type IIA fibres and the IIX-specific probes with type IIB fibres. The MyHC IIB mRNA probe hybridized only with a few histochemically defined type I fibres in the sample from the external oblique; in addition to this IIB message, these fibres also expressed RNAs for MyHC I, IIA and IIX. MyHC IIB RNA was abundantly expressed in histochemical and immunohistochemical type IIA fibres of the masseter, together with transcripts for IIA and in some cases IIX. No MyHC IIB protein was detected in fibres and extracts of either the external oblique or masseter by immunohistochemistry, immunoblotting and electrophoresis. Thus, IIB RNA, but not protein, was found in the fibres of two different human skeletal muscles. It is

  10. Geniohyoid muscle properties and myosin heavy chain composition are altered after short-term intermittent hypoxic exposure.

    PubMed

    Pae, Eung-Kwon; Wu, Jennifer; Nguyen, Daniel; Monti, Ryan; Harper, Ronald M

    2005-03-01

    Patients with obstructive sleep apnea (OSA) often exhibit fatigued or inefficient upper airway dilator and constrictor muscles; an upper airway dilator, the geniohyoid (GH) muscle, is a particular example. Intermittent hypoxia (IH) is a frequent concomitant of OSA, and it may trigger muscle fiber composition changes that are characteristic of a fatigable nature. We examined effects of short-term IH on diaphragmatic and GH muscle fiber composition and fatigue properties by exposing 24 rats to alternating 10.3% O(2)-balance N(2) and room air every 480 s (240 s duty cycle) for a total duration of 5, 10, 15, 20, or 30 h. Sternohyoid fiber composition was also examined. Control animals were exposed to room air on the same schedule. Single-fiber analyses showed that GH muscle fiber types changed completely from myosin heavy chain (MHC) type 2A to MHC type 2B after 10 h of exposure, and the conversion was maintained for at least 30 h. Sternohyoid muscle fibers showed a delayed transition from MHC type 2A/2B to MHC type 2B. In contrast, major fiber types of the diaphragm were not significantly altered. The GH muscles showed similar tension-frequency relationships in all groups, but an increased fatigability developed, proportional to the duration of IH treatment. We conclude that short-term IH exposure alters GH muscle composition and physical properties toward more fatigable, fast-twitch types and that it may account for the fatigable upper airway fiber types found in sleep-disturbed breathing. PMID:15557011

  11. Relationship between pork quality and characteristics of muscle fibers classified by the distribution of myosin heavy chain isoforms.

    PubMed

    Kim, Gap-Don; Ryu, Youn-Chul; Jeong, Jin-Yeon; Yang, Han-Sul; Joo, Seon-Tea

    2013-11-01

    A total of six fiber types, including four pure types (type I, IIA, IIX, and IIB) and two hybrid types (type IIAX and IIXB), were classified according to the expression of myosin heavy chain (MHC) isoforms by immunohistochemistry with MHC specific monoclonal antibodies. The comparison of the muscle fiber characteristics and pork quality between pork quality groups (DFD: dark, firm, and dry; PSE: pale, soft, and exudative; RFN: reddish pink, firm, and nonexudative; and RSE: reddish pink, soft, and exudative) classified by muscle pH, drip loss, and lightness was conducted and the relationship of myofiber characteristics to pork quality was investigated. The DFD group had the highest value of IIAX fiber density (P<0.05). The DFD group also showed the greatest fiber relative area of type I, IIA, and IIAX (P<0.05) whereas there were no significant differences in area composition for types I, IIA, and IIAX among the other groups including PSE, RFN, and RSE (P>0.05). The DFD group had the highest cross-sectional area (CSA) in types I, IIA, and IIX among the groups. The increase in density of type IIAX was related with the higher pH and the lower hue and drip loss. An increase in the fiber number composition of hybrid type IIXB increased the lightness and cooking loss and decreased sarcoplasmic protein solubility (SPS). Regarding fiber relative area, pure type I and IIA and hybrid type IIAX were greater in the DFD group and had lower lightness and drip loss. Hybrid type IIAX influences the desirability of the pork due to its association with low lightness and high pH and water-holding capacity (WHC). In contrast, type IIXB was related to poor quality pork, including pale color, low WHC in cooked meat, and low SPS. PMID:23989883

  12. Chronic hypoxia and VEGF differentially modulate abundance and organization of myosin heavy chain isoforms in fetal and adult ovine arteries.

    PubMed

    Hubbell, Margaret C; Semotiuk, Andrew J; Thorpe, Richard B; Adeoye, Olayemi O; Butler, Stacy M; Williams, James M; Khorram, Omid; Pearce, William J

    2012-11-15

    Chronic hypoxia increases vascular endothelial growth factor (VEGF) and thereby promotes angiogenesis. The present study explores the hypothesis that hypoxic increases in VEGF also remodel artery wall structure and contractility through phenotypic transformation of smooth muscle. Pregnant and nonpregnant ewes were maintained at sea level (normoxia) or 3,820 m (hypoxia) for the final 110 days of gestation. Common carotid arteries harvested from term fetal lambs and nonpregnant adults were denuded of endothelium and studied in vitro. Stretch-dependent contractile stresses were 32 and 77% of normoxic values in hypoxic fetal and adult arteries. Hypoxic hypocontractility was coupled with increased abundance of nonmuscle myosin heavy chain (NM-MHC) in fetal (+37%) and adult (+119%) arteries. Conversely, hypoxia decreased smooth muscle MHC (SM-MHC) abundance by 40% in fetal arteries but increased it 123% in adult arteries. Hypoxia decreased colocalization of NM-MHC with smooth muscle α-actin (SM-αA) in fetal arteries and decreased colocalization of SM-MHC with SM-αA in adult arteries. Organ culture with physiological concentrations (3 ng/ml) of VEGF-A(165) similarly depressed stretch-dependent stresses to 37 and 49% of control fetal and adult values. The VEGF receptor antagonist vatalanib ablated VEGF's effects in adult but not fetal arteries, suggesting age-dependent VEGF receptor signaling. VEGF replicated hypoxic decreases in colocalization of NM-MHC with SM-αA in fetal arteries and decreases in colocalization of SM-MHC with SM-αA in adult arteries. These results suggest that hypoxic increases in VEGF not only promote angiogenesis but may also help mediate hypoxic arterial remodeling through age-dependent changes in smooth muscle phenotype and contractility. PMID:22992677

  13. Myosin heavy chain composition of the human lateral pterygoid and digastric muscles in young adults and elderly.

    PubMed

    Monemi, M; Liu J-X; Thornell, L E; Eriksson, P O

    2000-05-01

    The myosin heavy chain (MyHC) content in different parts of, two jaw opening muscle, the human lateral pterygoid and the digastric muscles of five young adult and five elderly subjects (mean age 22 and 73 years, respectively) was determined, using gel electrophoresis and immunohistochemical methods. The lateral pterygoid of both young and elderly contained predominantly slow MyHC, and fast A MyHC was the major fast isoform. In contrast, the digastric was composed of slow, fast A and fast X MyHCs in about equal proportions in both age groups. About half of the lateral pterygoid fibres contained mixtures of slow and fast MyHCs, often together with alpha-cardiac MyHC. In the digastric, co-existence of slow and fast MyHCs was rare, and alpha-cardiac MyHC was lacking. On the other hand, co-expression of fast A and fast X MyHCs was found more often in the digastric than in the lateral pterygoid. In both age groups about half of the digastric IIB fibres contained solely fast X MyHC. In the lateral pterygoid, type IIB fibres with pure fast X MyHC was found in only one subject. The lateral pterygoid in elderly showed a significant amount of fibres with solely fast A MyHC, which were occasionally found in young adults. In the digastric, no significant differences were found between young and elderly, although the muscles of elderly contained lower mean value of slow MyHC, as compared to that of young muscles. It is concluded that the lateral pterygoid and the digastric muscles differ not only in the MyHC composition but also in modifications of the MyHC phenotypes during aging, suggesting that they have separate roles in jaw opening function. PMID:11032341

  14. Histone Deacetylase 3 (HDAC3)-dependent Reversible Lysine Acetylation of Cardiac Myosin Heavy Chain Isoforms Modulates Their Enzymatic and Motor Activity*

    PubMed Central

    Samant, Sadhana A.; Pillai, Vinodkumar B.; Sundaresan, Nagalingam R.; Shroff, Sanjeev G.; Gupta, Mahesh P.

    2015-01-01

    Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, p300/CREB-binding protein-associated factor, associate with cardiac sarcomeres and that a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to A-band of sarcomeres and capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the Km for the actin-activated ATPase activity of MHC isoforms. By in vitro motility assay, we found that lysine acetylation increased the actin-sliding velocity of α-myosin by 20% and β-myosin by 36% compared with their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli independently of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms. PMID:25911107

  15. Histone Deacetylase 3 (HDAC3)-dependent Reversible Lysine Acetylation of Cardiac Myosin Heavy Chain Isoforms Modulates Their Enzymatic and Motor Activity.

    PubMed

    Samant, Sadhana A; Pillai, Vinodkumar B; Sundaresan, Nagalingam R; Shroff, Sanjeev G; Gupta, Mahesh P

    2015-06-19

    Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, p300/CREB-binding protein-associated factor, associate with cardiac sarcomeres and that a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to A-band of sarcomeres and capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the Km for the actin-activated ATPase activity of MHC isoforms. By in vitro motility assay, we found that lysine acetylation increased the actin-sliding velocity of α-myosin by 20% and β-myosin by 36% compared with their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli independently of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms. PMID:25911107

  16. Single Muscle Immobilization Decreases Single-Fibre Myosin Heavy Chain Polymorphism: Possible Involvement of p38 and JNK MAP Kinases

    PubMed Central

    Derbré, Frédéric; Droguet, Mickaël; Léon, Karelle; Troadec, Samuel; Pennec, Jean-Pierre; Giroux-Metges, Marie-Agnès; Rannou, Fabrice

    2016-01-01

    Purpose Muscle contractile phenotype is affected during immobilization. Myosin heavy chain (MHC) isoforms are the major determinant of the muscle contractile phenotype. We therefore sought to evaluate the effects of muscle immobilization on both the MHC composition at single-fibre level and the mitogen-activated protein kinases (MAPK), a family of intracellular signaling pathways involved in the stress-induced muscle plasticity. Methods The distal tendon of female Wistar rat Peroneus Longus (PL) was cut and fixed to the adjacent bone at neutral muscle length. Four weeks after the surgery, immobilized and contralateral PL were dissociated and the isolated fibres were sampled to determine MHC composition. Protein kinase 38 (p38), extracellular signal-regulated kinases (ERK1/2), and c-Jun- NH2-terminal kinase (JNK) phosphorylations were measured in 6- and 15-day immobilized and contralateral PL. Results MHC distribution in immobilized PL was as follows: I = 0%, IIa = 11.8 ± 2.8%, IIx = 53.0 ± 6.1%, IIb = 35.3 ± 7.3% and I = 6.1 ± 3.9%, IIa = 22.1 ± 3.4%, IIx = 46.6 ± 4.5%, IIb = 25.2 ± 6.6% in contralateral muscle. The MHC composition in immobilized muscle is consistent with a faster contractile phenotype according to the Hill’s model of the force-velocity relationship. Immobilized and contralateral muscles displayed a polymorphism index of 31.1% (95% CI 26.1–36.0) and 39.3% (95% CI 37.0–41.5), respectively. Significant increases in p38 and JNK phosphorylation were observed following 6 and 15 days of immobilization. Conclusions Single muscle immobilization at neutral length induces a shift of MHC composition toward a faster contractile phenotype and decreases the polymorphic profile of single fibres. Activation of p38 and JNK could be a potential mechanism involved in these contractile phenotype modifications during muscle immobilization. PMID:27383612

  17. Force-velocity relations and myosin heavy chain isoform compositions of skinned fibres from rat skeletal muscle.

    PubMed Central

    Bottinelli, R; Schiaffino, S; Reggiani, C

    1991-01-01

    1. This study was performed to assess whether muscle contractile properties are related to the presence of specific myosin heavy chain (MHC) isoforms. 2. Force-velocity relations and MHC isoform composition were determined in seventy-four single skinned muscle fibres from rat soleus, extensor digitorum longus and plantaris muscles. 3. Four groups of fibres were identified according to their MHC isoform composition determined by monoclonal antibodies: type 1 (slow), and types 2A, 2B and 2X (fast). 4. With respect to maximum velocity of shortening (V0), the fibres formed a continuum between 0.35 and 2.84 L/s (muscle lengths per second) at 12 degrees C. V0 in type 1 fibres (slow fibres) was between 0.35 and 0.95 L/s (0.639 +/- 0.038 L/s; mean +/- S.E. of mean). V0 in type 2 fibres (fast fibres) was consistently higher than 0.91 L/s. Ranges of V0 in the three fast fibre types mostly overlapped. Type 2A and 2X fibres had similar mean V0 values (1.396 +/- 0.084 and 1.451 +/- 0.066 L/s respectively); type 2B fibres showed a higher mean V0 value (1.800 +/- 0.109 L/s) than type 2A and 2X fibres. 5. Mean values of a/P0, an index of the curvature of force-velocity relations, allowed us to identify two groups of fibres: a high curvature group comprised of type 1 (mean a/P0, 0.066 +/- 0.007) and 2A (0.066 +/- 0.024) fibres and a low curvature group comprised of type 2B (0.113 +/- 0.013) and 2X (0.132 +/- 0.008) fibres. 6. Maximal power output was lower in slow fibres than in fast fibres, and among fast fibres it was lower in type 2A fibres than in type 2X and 2B. 7. Force per unit cross-sectional area was less in slow fibres than in fast fibres. There was no relation between fibre type and cross-sectional area. 8. The results suggest that MHC composition is just one of the determinants of shortening velocity and of other muscle contractile properties. Images Fig. 3 PMID:1890654

  18. Association between myosin heavy chain protein isoforms and intramuscular anabolic signaling following resistance exercise in trained men.

    PubMed

    Gonzalez, Adam M; Hoffman, Jay R; Townsend, Jeremy R; Jajtner, Adam R; Wells, Adam J; Beyer, Kyle S; Willoughby, Darryn S; Oliveira, Leonardo P; Fukuda, David H; Fragala, Maren S; Stout, Jeffrey R

    2015-01-01

    Resistance exercise stimulates an increase in muscle protein synthesis regulated by intracellular anabolic signaling molecules in a mammalian/mechanistic target of rapamycin (mTOR)-dependent pathway. The purpose of this study was to investigate acute anabolic signaling responses in experienced, resistance-trained men, and to examine the association between myosin heavy chain (MHC) isoform composition and the magnitude of anabolic signaling. Eight resistance-trained men (24.9 ± 4.3 years; 91.2 ± 12.4 kg; 176.7 ± 8.0 cm; 13.3 ± 3.9 body fat %) performed a whole body, high-volume resistance exercise protocol (REX) and a control protocol (CTL) in a balanced, randomized order. Participants were provided a standardized breakfast, recovery drink, and meal during each protocol. Fine needle muscle biopsies were completed at baseline (BL), 2 h (2H) and 6 h post-exercise (6H). BL biopsies were analyzed for MHC isoform composition. Phosphorylation of proteins specific to the Akt/mTOR signaling pathway and MHC mRNA expression was quantified. Phosphorylation of p70S6k was significantly greater in REX compared to CTL at 2H (P = 0.04). MHC mRNA expression and other targets in the Akt/mTOR pathway were not significantly influenced by REX. The percentage of type IIX isoform was inversely correlated (P < 0.05) with type I and type IIA MHC mRNA expression (r = -0.69 to -0.93). Maximal strength was also observed to be inversely correlated (P < 0.05) with Type I and Type IIA MHC mRNA expression (r = -0.75 to -0.77) and p70S6k phosphorylation (r = -0.75). Results indicate that activation of p70S6k occurs within 2-h following REX in experienced, resistance-trained men. Further, results also suggest that highly trained, stronger individuals have an attenuated acute anabolic response. PMID:25626869

  19. Association between myosin heavy chain protein isoforms and intramuscular anabolic signaling following resistance exercise in trained men

    PubMed Central

    Gonzalez, Adam M.; Hoffman, Jay R.; Townsend, Jeremy R.; Jajtner, Adam R.; Wells, Adam J.; Beyer, Kyle S.; Willoughby, Darryn S.; Oliveira, Leonardo P.; Fukuda, David H.; Fragala, Maren S.; Stout, Jeffrey R.

    2015-01-01

    Abstract Resistance exercise stimulates an increase in muscle protein synthesis regulated by intracellular anabolic signaling molecules in a mammalian/mechanistic target of rapamycin (mTOR)‐dependent pathway. The purpose of this study was to investigate acute anabolic signaling responses in experienced, resistance‐trained men, and to examine the association between myosin heavy chain (MHC) isoform composition and the magnitude of anabolic signaling. Eight resistance‐trained men (24.9 ± 4.3 years; 91.2 ± 12.4 kg; 176.7 ± 8.0 cm; 13.3 ± 3.9 body fat %) performed a whole body, high‐volume resistance exercise protocol (REX) and a control protocol (CTL) in a balanced, randomized order. Participants were provided a standardized breakfast, recovery drink, and meal during each protocol. Fine needle muscle biopsies were completed at baseline (BL), 2 h (2H) and 6 h post‐exercise (6H). BL biopsies were analyzed for MHC isoform composition. Phosphorylation of proteins specific to the Akt/mTOR signaling pathway and MHC mRNA expression was quantified. Phosphorylation of p70S6k was significantly greater in REX compared to CTL at 2H (P = 0.04). MHC mRNA expression and other targets in the Akt/mTOR pathway were not significantly influenced by REX. The percentage of type IIX isoform was inversely correlated (P < 0.05) with type I and type IIA MHC mRNA expression (r = −0.69 to −0.93). Maximal strength was also observed to be inversely correlated (P < 0.05) with Type I and Type IIA MHC mRNA expression (r = −0.75 to −0.77) and p70S6k phosphorylation (r = −0.75). Results indicate that activation of p70S6k occurs within 2‐h following REX in experienced, resistance‐trained men. Further, results also suggest that highly trained, stronger individuals have an attenuated acute anabolic response. PMID:25626869

  20. Evidence for myoblast-extrinsic regulation of slow myosin heavy chain expression during muscle fiber formation in embryonic development.

    PubMed

    Cho, M; Webster, S G; Blau, H M

    1993-05-01

    Vertebrate muscles are composed of an array of diverse fast and slow fiber types with different contractile properties. Differences among fibers in fast and slow MyHC expression could be due to extrinsic factors that act on the differentiated myofibers. Alternatively, the mononucleate myoblasts that fuse to form multinucleated muscle fibers could differ intrinsically due to lineage. To distinguish between these possibilities, we determined whether the changes in proportion of slow fibers were attributable to inherent differences in myoblasts. The proportion of fibers expressing slow myosin heavy chain (MyHC) was found to change markedly with time during embryonic and fetal human limb development. During the first trimester, a maximum of 75% of fibers expressed slow MyHC. Thereafter, new fibers formed which did not express this MyHC, so that the proportion of fibers expressing slow MyHC dropped to approximately 3% of the total by midgestation. Several weeks later, a subset of the new fibers began to express slow MyHC and from week 30 of gestation through adulthood, approximately 50% of fibers were slow. However, each myoblast clone (n = 2,119) derived from muscle tissues at six stages of human development (weeks 7, 9, 16, and 22 of gestation, 2 mo after birth and adult) expressed slow MyHC upon differentiation. We conclude from these results that the control of slow MyHC expression in vivo during muscle fiber formation in embryonic development is largely extrinsic to the myoblast. By contrast, human myoblast clones from the same samples differed in their expression of embryonic and neonatal MyHCs, in agreement with studies in other species, and this difference was shown to be stably heritable. Even after 25 population doublings in tissue culture, embryonic stage myoblasts did not give rise to myoblasts capable of expressing MyHCs typical of neonatal stages, indicating that stage-specific differences are not under the control of a division dependent mechanism, or

  1. Evidence for myoblast-extrinsic regulation of slow myosin heavy chain expression during muscle fiber formation in embryonic development

    PubMed Central

    1993-01-01

    Vertebrate muscles are composed of an array of diverse fast and slow fiber types with different contractile properties. Differences among fibers in fast and slow MyHC expression could be due to extrinsic factors that act on the differentiated myofibers. Alternatively, the mononucleate myoblasts that fuse to form multinucleated muscle fibers could differ intrinsically due to lineage. To distinguish between these possibilities, we determined whether the changes in proportion of slow fibers were attributable to inherent differences in myoblasts. The proportion of fibers expressing slow myosin heavy chain (MyHC) was found to change markedly with time during embryonic and fetal human limb development. During the first trimester, a maximum of 75% of fibers expressed slow MyHC. Thereafter, new fibers formed which did not express this MyHC, so that the proportion of fibers expressing slow MyHC dropped to approximately 3% of the total by midgestation. Several weeks later, a subset of the new fibers began to express slow MyHC and from week 30 of gestation through adulthood, approximately 50% of fibers were slow. However, each myoblast clone (n = 2,119) derived from muscle tissues at six stages of human development (weeks 7, 9, 16, and 22 of gestation, 2 mo after birth and adult) expressed slow MyHC upon differentiation. We conclude from these results that the control of slow MyHC expression in vivo during muscle fiber formation in embryonic development is largely extrinsic to the myoblast. By contrast, human myoblast clones from the same samples differed in their expression of embryonic and neonatal MyHCs, in agreement with studies in other species, and this difference was shown to be stably heritable. Even after 25 population doublings in tissue culture, embryonic stage myoblasts did not give rise to myoblasts capable of expressing MyHCs typical of neonatal stages, indicating that stage-specific differences are not under the control of a division dependent mechanism, or

  2. Expression profiles of myostatin, myogenin, and Myosin heavy chain in skeletal muscles of two rabbit breeds differing in growth rate.

    PubMed

    Kuang, Liangde; Xie, Xiaohong; Zhang, Xiangyu; Lei, Min; Li, Congyan; Ren, Yongjun; Zheng, Jie; Guo, Zhiqiang; Zhang, Cuixia; Yang, Chao; Zheng, Yucai

    2014-01-01

    The purpose of the present study was to compare mRNA levels of myostatin (MSTN), myogenin (MyoG), and fiber type compositions in terms of myosin heavy chain (MyHC) in skeletal muscles of two rabbit breeds with different body sizes and growth rates. Longissimus dorsi and biceps femoris muscles of 16 Californian rabbits (CW) and 16 Germany great line of ZIKA rabbits (GZ) were collected at the ages of 35d and 84d (slaughter age). The results showed that the live weights of GZ rabbits of 35d and 84d old were approximately 36% and 26% greater than those of CW rabbits, respectively. Quantitative real-time PCR analysis revealed that at the age of 84d GZ rabbits contained significantly lower MSTN mRNA level and higher MyoG mRNA level in both longissimus dorsi and biceps femoris muscles than CW rabbits, and mRNA levels of MSTN and MyoG exhibited opposite changes from the age of 35d to 84d, suggesting that GZ rabbits were subjected to less growth inhibition from MSTN at slaughter age, which occurred most possibly in skeletal muscles. Four types of fiber were identified by real-time PCR in rabbit muscles, with MyHC-1 and MyHC-2D, MyHC-2B were the major types in biceps femoris and longissimus dorsi muscles, respectively. At the age of 84d, GZ rabbits contained greater proportion of MyHC-1 and decreased proportion of MyHC-2D and decreased lactate dehydrogenase activity in biceps femoris than CW rabbits, and the results were exactly opposite in longissimus dorsi, suggesting that GZ rabbits show higher oxidative capacity in biceps femoris muscle than CW rabbits. In conclusion, the trends of mRNA levels of MSTN and fiber types in GZ rabbits' skeletal muscles might be consistent with the putative fast growth characteristic of GZ rabbits compared to CW rabbits. PMID:24813217

  3. Responses of Myosin Heavy Chain Phenotypes and Gene Expressions in Neck Muscle to Micro- an Hyper-Gravity in Mice

    NASA Astrophysics Data System (ADS)

    Ohira, Tomotaka; Ohira, Takashi; Kawano, F.; Shibaguchi, T.; Okabe, H.; Ohno, Y.; Nakai, N.; Ochiai, T.; Goto, K.; Ohira, Y.

    2013-02-01

    Neck muscles are known to play important roles in the maintenance of head posture against gravity. However, it is not known how the properties of neck muscle are influenced by gravity. Therefore, the current study was performed to investigate the responses of neck muscle (rhomboideus capitis) in mice to inhibition of gravity and/or increase to 2-G for 3 months to test the hypothesis that the properties of neck muscles are regulated in response to the level of mechanical load applied by the gravitational load. Three male wild type C57BL/10J mice (8 weeks old) were launched by space shuttle Discovery (STS-128) and housed in Japanese Experimental Module “KIBO” on the International Space Station in mouse drawer system (MDS) project, which was organized by Italian Space Agency. Only 1 mouse returned to the Earth alive after 3 months by space shuttle Atlantis (STS-129). Neck muscles were sampled from both sides within 3 hours after landing. Cage and laboratory control experiments were also performed on the ground. Further, 3-month ground-based control experiments were performed with 6 groups, i.e. pre-experiment, 3-month hindlimb suspension, 2-G exposure by using animal centrifuge, and vivarium control (n=5 each group). Five mice were allowed to recover from hindlimb suspension (including 5 cage control) for 3 months in the cage. Neck muscles were sampled bilaterally before and after 3-month suspension and 2-G exposure, and at the end of 3-month ambulation recovery. Spaceflight-associated shift of myosin heavy chain phenotype from type I to II and atrophy of type I fibers were observed. In response to spaceflight, 17 genes were up-regulated and 13 genes were down-regulated vs. those in the laboratory control. Expression of 6 genes were up-regulated and that of 88 genes were down-regulated by 3-month exposure to 2-G vs. the age-matched cage control. In response to chronic hindlimb suspension, 4 and 20 genes were up- or down-regulated. Further, 98 genes responded

  4. Dietary cholecalciferol regulates the recruitment and growth of skeletal muscle fibers and the expressions of myogenic regulatory factors and the myosin heavy chain in European sea bass larvae.

    PubMed

    Alami-Durante, Hélène; Cluzeaud, Marianne; Bazin, Didier; Mazurais, David; Zambonino-Infante, José L

    2011-12-01

    The aim of this study was to determine whether dietary cholecalciferol affects the recruitment and growth of axial skeletal muscle fibers in first-feeding European sea bass. Larvae were fed diets containing 0.28 (VD-L, low dose), 0.69 (VD-C, control dose), or 3.00 (VD-H, high dose) mg cholecalciferol/kg from 9 to 44 d posthatching (dph). Larvae were sampled at 44 dph for quantification of somatic growth, muscle growth, and muscle growth dynamics and at 22 and 44 dph for the relative quantification of transcripts encoded by genes involved in myogenesis, cell proliferation, and muscle structure. The weight increase of the VD-L-fed larvae was less than that of the VD-H-fed group, whereas that of VD-C-fed larvae was intermediate. The level of expression of genes involved in cell proliferation (PCNA) and early myogenesis (Myf5) decreased between 22 and 44 dph, whereas that of the myogenic determination factor MyoD1 and that of genes involved in muscle structure and function (myosin heavy chain, myosin light chains 2 and 3) increased. Dietary cholecalciferol regulated Myf5, MyoD1, myogenin, and myosin heavy chain gene expression, with a gene-specific shape of response. The maximum hypertrophy of white muscle fibers was higher in larvae fed the VD-C and VD-H diets than in larvae fed the VD-L diet. White muscle hyperplasia was highly stimulated in VD-H-fed larvae compared to VD-L- and VD-C-fed ones. These findings demonstrate a dietary cholecalciferol effect on skeletal muscle growth mechanisms of a Teleost species. PMID:22013200

  5. MicroRNA-23a reduces slow myosin heavy chain isoforms composition through myocyte enhancer factor 2C (MEF2C) and potentially influences meat quality.

    PubMed

    Shen, Linyuan; Chen, Lei; Zhang, Shunhua; Zhang, Yi; Wang, Jingyong; Zhu, Li

    2016-06-01

    MicroRNAs (miRNAs) are non-coding small RNAs that participate in the regulation of a variety of biological processes. Muscle fiber types were very important to meat quality traits, however, the molecular mechanism by which miRNAs regulate the muscle fiber type composition is not fully understood. The aim of this study was to investigate whether miRNA-23a can affect muscle fiber type composition. Luciferase reporter assays proved that miRNA-23a directly targets the 3' untranslated region (UTRs) of MEF2c. Overexpression of miRNA-23a significantly suppressed the expression of MEF2c both in mRNA and protein levels, thus caused down-regulation of the expression of some key downstream genes of MEF2c (PGC1-α, NRF1 and mtTFA). More interestingly, overexpression of miRNA-23a significantly restrained the myogenic differentiation and decreased the ratio of slow myosin heavy chain in myoblasts (p<0.05). Our findings hinted a novel role of miRNA-23a in the epigenetic regulation of meat quality via decreasing the ratio of slow myosin heavy chain isoforms. PMID:26897085

  6. Four things to know about myosin light chains as reporters for non-muscle myosin-2 dynamics in live cells.

    PubMed

    Heissler, Sarah M; Sellers, James R

    2015-02-01

    The interplay between non-muscle myosins-2 and filamentous actin results in cytoplasmic contractility which is essential for eukaryotic life. Concomitantly, there is tremendous interest in elucidating the physiological function and temporal localization of non-muscle myosin-2 in cells. A commonly used method to study the function and localization of non-muscle myosin-2 is to overexpress a fluorescent protein (FP)-tagged version of the regulatory light chain (RLC) which binds to the myosin-2 heavy chain by mass action. Caveats about this approach include findings from recent studies indicating that the RLC does not bind exclusively to the non-muscle myosin-2 heavy chain. Rather, it can also associate with the myosin heavy chains of several other classes as well as other targets than myosin. In addition, the presence of the FP moiety may compromise myosin's enzymatic and mechanical performance. This and other factors to be discussed in this commentary raise questions about the possible complications in using FP-RLC as a marker for the dynamic localization and regulatory aspects of non-muscle myosin-2 motor functions in cell biological experiments. PMID:25712372

  7. Immunocytochemistry for the heavy chain of the non-muscle myosin IIA as a diagnostic tool for MYH9-related disorders.

    PubMed

    Pecci, Alessandro; Noris, Patrizia; Invernizzi, Rosangela; Savoia, Anna; Seri, Marco; Ghiggeri, Gian Marco; Sartore, Saverio; Gangarossa, Simone; Bizzaro, Nicola; Balduini, Carlo L

    2002-04-01

    May-Hegglin anomaly (MHA), Sebastian syndrome (SBS) and Fechtner syndrome (FTNS) are autosomal-dominant macrothrombocytopenias with Döhle-like leucocyte inclusions. These diseases are due to mutations of the MHY9 gene, encoding the heavy chain of non-muscle myosin IIA (NMMHC-A). We investigated the NMMHC-A localization in blood cells from eight MHA, SBS or FTNS patients with known MYH9 mutations. All the patients showed an altered localization of NMMHC-A in granulocytes and platelets, suggesting that Döhle-like bodies are due to the aggregation of NMMHC-A in the cytoplasm. Therefore, immunocytochemistry for NMMHC-A is a simple and sensitive method to detect pathological phenotypes of granulocytes and platelets in the diagnosis of MYH9-related disorders. PMID:11918549

  8. Structure of the Dictyostelium Myosin-II Heavy Chain Kinase A (MHCK-A) α-kinase domain apoenzyme reveals a novel autoinhibited conformation.

    PubMed

    Ye, Qilu; Yang, Yidai; van Staalduinen, Laura; Crawley, Scott William; Liu, Linda; Brennan, Stephanie; Côté, Graham P; Jia, Zongchao

    2016-01-01

    The α-kinases are a family of a typical protein kinases present in organisms ranging from protozoa to mammals. Here we report an autoinhibited conformation for the α-kinase domain of Dictyostelium myosin-II heavy chain kinase A (MHCK-A) in which nucleotide binding to the catalytic cleft, located at the interface between an N-terminal and C-terminal lobe, is sterically blocked by the side chain of a conserved arginine residue (Arg592). Previous α-kinase structures have shown that an invariant catalytic aspartic acid residue (Asp766) is phosphorylated. Unexpectedly, in the autoinhibited conformation the phosphoryl group is transferred to the adjacent Asp663, creating an interaction network that stabilizes the autoinhibited state. The results suggest that Asp766 phosphorylation may play both catalytic and regulatory roles. The autoinhibited structure also provides the first view of a phosphothreonine residue docked into the phospho-specific allosteric binding site (Pi-pocket) in the C-lobe of the α-kinase domain. PMID:27211275

  9. Structure of the Dictyostelium Myosin-II Heavy Chain Kinase A (MHCK-A) α-kinase domain apoenzyme reveals a novel autoinhibited conformation

    PubMed Central

    Ye, Qilu; Yang, Yidai; van Staalduinen, Laura; Crawley, Scott William; Liu, Linda; Brennan, Stephanie; Côté, Graham P.; Jia, Zongchao

    2016-01-01

    The α-kinases are a family of a typical protein kinases present in organisms ranging from protozoa to mammals. Here we report an autoinhibited conformation for the α-kinase domain of Dictyostelium myosin-II heavy chain kinase A (MHCK-A) in which nucleotide binding to the catalytic cleft, located at the interface between an N-terminal and C-terminal lobe, is sterically blocked by the side chain of a conserved arginine residue (Arg592). Previous α-kinase structures have shown that an invariant catalytic aspartic acid residue (Asp766) is phosphorylated. Unexpectedly, in the autoinhibited conformation the phosphoryl group is transferred to the adjacent Asp663, creating an interaction network that stabilizes the autoinhibited state. The results suggest that Asp766 phosphorylation may play both catalytic and regulatory roles. The autoinhibited structure also provides the first view of a phosphothreonine residue docked into the phospho-specific allosteric binding site (Pi-pocket) in the C-lobe of the α-kinase domain. PMID:27211275

  10. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    SciTech Connect

    Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.; Burghardt, Thomas P.; Ajtai, Katalin

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant

  11. Temporal embryonic transcription of chicken fast skeletal myosin heavy chain isoforms in the single comb white leghorn.

    PubMed

    Griffin, J; St-Pierre, N; Lilburn, M S; Wick, M

    2016-05-01

    There are numerous factors that can significantly influence embryonic development in poultry and thus make simple days of incubation (chronological age) a less than perfect metric for studying embryonic physiology. The developmental fast skeletal muscle myosin (MyHC), the predominant protein in the Pectoralis major (PM), is temporally expressed as a cadre of highly specific developmental isoforms. In the study described herein, a novel molecular technology (NanoString) was used to characterize the myosin isoform transcriptional patterns in the PM of Single Comb White Leghorn (SCWL) embryos. NanoString technology is based on quantitative analysis of the transcriptome through digital detection and quantification of target mRNA transcripts. Total RNA was isolated and gene transcription quantified using NanoString in embryonic muscle samples collected daily from 6 through 19 days of incubation. Data were analyzed using the LOESS smoothing function at a 95% confidence level. The temporal transcription of MyHC isoforms obtained in this study was consistent with the literature at higher specificity and resolution, thus validating NanoString for use in gene transcription analyses. The results support a hypothesis that the transcription patterns of the embryonic MyHC isoforms may be used as molecular clocks to further investigate the developmental relationships underlying embryonic fast skeletal muscle growth and development. PMID:26908894

  12. Temporal embryonic transcription of chicken fast skeletal myosin heavy chain isoforms in the single comb white leghorn

    PubMed Central

    Griffin, J.; St-Pierre, N.; Lilburn, M. S.; Wick, M.

    2016-01-01

    There are numerous factors that can significantly influence embryonic development in poultry and thus make simple days of incubation (chronological age) a less than perfect metric for studying embryonic physiology. The developmental fast skeletal muscle myosin (MyHC), the predominant protein in the Pectoralis major (PM), is temporally expressed as a cadre of highly specific developmental isoforms. In the study described herein, a novel molecular technology (NanoString) was used to characterize the myosin isoform transcriptional patterns in the PM of Single Comb White Leghorn (SCWL) embryos. NanoString technology is based on quantitative analysis of the transcriptome through digital detection and quantification of target mRNA transcripts. Total RNA was isolated and gene transcription quantified using NanoString in embryonic muscle samples collected daily from 6 through 19 days of incubation. Data were analyzed using the LOESS smoothing function at a 95% confidence level. The temporal transcription of MyHC isoforms obtained in this study was consistent with the literature at higher specificity and resolution, thus validating NanoString for use in gene transcription analyses. The results support a hypothesis that the transcription patterns of the embryonic MyHC isoforms may be used as molecular clocks to further investigate the developmental relationships underlying embryonic fast skeletal muscle growth and development. PMID:26908894

  13. Membrane-bound Dictyostelium myosin heavy chain kinase: a developmentally regulated substrate-specific member of the protein kinase C family.

    PubMed Central

    Ravid, S; Spudich, J A

    1992-01-01

    A cDNA clone corresponding to the Dictyostelium myosin heavy chain kinase (MHCK) gene was isolated using antibodies specific to the purified enzyme. Sequence analysis of the cDNA revealed that the Dictyostelium MHCK possesses all of the domains characteristic of members of the protein kinase C family. The amino-terminal region of the MHCK contains the cysteine-rich motif with an internal duplication that is present in all known protein kinase C species. This domain precedes sequences that are highly homologous to protein kinase catalytic domains. The carboxyl-terminal region contains a cluster of 23 serine and threonine residues that may represent the autophosphorylation domain of the Dictyostelium MHCK. These results, along with previous studies that indicate that this enzyme has very restrictive substrate specificity, incorporates approximately 20 mol of phosphate per mol of kinase through an autophosphorylation reaction, and is expressed only during development, suggest that the Dictyostelium MHCK is a distinct member of the protein kinase C family and imply that this kinase family, which may include members with very specific cellular functions, may be even more heterogeneous than previously thought. Images PMID:1321427

  14. Pilot study identifying myosin heavy chain 7, desmin, insulin-like growth factor 7, and annexin A2 as circulating biomarkers of human heart failure

    PubMed Central

    Chugh, S.; Ouzounian, M.; Lu, Z.; Mohamed, S.; Li, W.; Bousette, N.; Liu, P.P.; Gramolini, A.O.

    2013-01-01

    In depth proteomic analyses offer a systematic way to investigate protein alterations in disease and, as such, can be a powerful tool for the identification of novel biomarkers. Here, we analyzed proteomic data from a transgenic mouse model with cardiac-specific overexpression of activated calcineurin (CnA), which results in severe cardiac hypertrophy. We applied statistically filtering and false discovery rate correction methods to identify 52 proteins that were significantly different in the CnA hearts compared to controls. Subsequent informatic analysis consisted of comparison of these 52 CnA proteins to another proteomic dataset of heart failure, three available independent microarray datasets, and correlation of their expression with the human plasma and urine proteome. Following this filtering strategy, four proteins passed these selection criteria including: myosin heavy chain 7, insulin-like growth factor-binding protein 7, annexin A2, and desmin. We assessed expression levels of these proteins in mouse plasma by immunoblotting, and observed significantly different levels of expression between healthy and failing mice for all 4 proteins. We verified antibody cross reactivity by examining human cardiac explant tissue by immunoblotting. Finally, we assessed protein levels in plasma samples obtained from 4 unaffected and 4 heart failure patients and demonstrated that all four proteins increased between 2-fold and 150-fold in heart failure. We conclude that MYH7, IGFBP7, ANXA2, and DESM are all excellent candidate plasma biomarkers of heart failure in mouse and human. PMID:23713052

  15. In vivo regulation of the beta-myosin heavy chain gene in soleus muscle of suspended and weight-bearing rats

    NASA Technical Reports Server (NTRS)

    Giger, J. M.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

    2000-01-01

    In the weight-bearing hindlimb soleus muscle of the rat, approximately 90% of muscle fibers express the beta-myosin heavy chain (beta-MHC) isoform protein. Hindlimb suspension (HS) causes the MHC isoform population to shift from beta toward the fast MHC isoforms. Our aim was to establish a model to test the hypothesis that this shift in expression is transcriptionally regulated through specific cis elements of the beta-MHC promoter. With the use of a direct gene transfer approach, we determined the activity of different length beta-MHC promoter fragments, linked to a firefly luciferase reporter gene, in soleus muscle of control and HS rats. In weight-bearing rats, the relative luciferase activity of the longest beta-promoter fragment (-3500 bp) was threefold higher than the shorter promoter constructs, which suggests that an enhancer sequence is present in the upstream promoter region. After 1 wk of HS, the reporter activities of the -3500-, -914-, and -408-bp promoter constructs were significantly reduced ( approximately 40%), compared with the control muscles. However, using the -215-bp construct, no differences in promoter activity were observed between HS and control muscles, which indicates that the response to HS in the rodent appears to be regulated within the -408 and -215 bp of the promoter.

  16. Muscle-Specific Myosin Heavy Chain Shifts in Response to a Long-Term High Fat/High Sugar Diet and Resveratrol Treatment in Nonhuman Primates

    PubMed Central

    Hyatt, Jon-Philippe K.; Nguyen, Lisa; Hall, Allison E.; Huber, Ashley M.; Kocan, Jessica C.; Mattison, Julie A.; de Cabo, Rafael; LaRocque, Jeannine R.; Talmadge, Robert J.

    2016-01-01

    Shifts in myosin heavy chain (MHC) expression within skeletal muscle can be induced by a host of stimuli including, but not limited to, physical activity, alterations in neural activity, aging, and diet or obesity. Here, we hypothesized that both age and a long-term (2 year) high fat/high sugar diet (HFS) would induce a slow to fast MHC shift within the plantaris, soleus, and extensor digitorum longus (EDL) muscles from rhesus monkeys. Furthermore, we tested whether supplementation with resveratrol, a naturally occurring compound that has been attributed with augmenting aerobic potential through mitochondrial proliferation, would counteract any diet-induced MHC changes by promoting a fast to slow isoform switch. In general, we found that MHC isoforms were not altered by aging during mid-life. The HFS diet had the largest impact within the soleus muscle where the greatest slow to fast isoform shifts were observed in both mRNA and protein indicators. As expected, long-term resveratrol treatment counteracted, or blunted, these diet-induced shifts within the soleus muscle. The plantaris muscle also demonstrated a fast-to-slow phenotypic response to resveratrol treatment. In conclusion, diet or resveratrol treatment impacts skeletal muscle phenotype in a muscle-specific manner and resveratrol supplementation may be one approach for promoting the fatigue-resistant MHC (type I) isoform especially if its expression is blunted as a result of a long-term high fat/sugar diet. PMID:26973542

  17. Molecular cloning and mRNA expression analysis of myosin heavy chain (MyHC) from fast skeletal muscle of grass carp, Ctenopharyngodon idella

    NASA Astrophysics Data System (ADS)

    Chu, Wuying; Fu, Guihong; Bing, Shiyu; Meng, Tao; Zhou, Ruixue; Cheng, Jia; Zhao, Falan; Zhang, Hongfang; Zhang, Jianshe

    2010-03-01

    The myosin heavy chain (MyHC) is one of the major structural and contracting proteins of muscle. We have isolated the cDNA clone encoding MyHC of the grass carp, Ctenopharyngodon idella. The sequence comprises 5 934 bp, including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues. The deduced amino acid sequence showed 69% homology to rabbit fast skeletal MyHC and 73%-76% homology to the MyHCs from the mandarin fish, walleye pollack, white croaker, chum salmon, and carp. The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%-80% homology to the corresponding regions of other fish MyHCs. The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR. The MyHC gene showed the highest expression in the muscles compared with the kidney, spleen and intestine. Developmentally, there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage. The highest expression was detected in hatching larva. Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.

  18. Muscle fiber type specific activation of the slow myosin heavy chain 2 promoter by a non-canonical E-box.

    PubMed

    Weimer, Kristina; DiMario, Joseph X

    2016-01-22

    Different mechanisms control skeletal muscle fiber type gene expression at specific times in vertebrate development. Embryonic myogenesis leading to formation of primary muscle fibers in avian species is largely directed by myoblast cell commitment to the formation of diverse fiber types. In contrast, development of different secondary fiber types during fetal myogenesis is partly determined by neural influences. In both primary and secondary chicken muscle fibers, differential expression of the slow myosin heavy chain 2 (MyHC2) gene distinguishes fast from fast/slow muscle fibers. This study focused on the transcriptional regulation of the slow MyHC2 gene in primary myotubes formed from distinct fast/slow and fast myogenic cell lineages. Promoter deletion analyses identified a discrete 86 bp promoter segment that conferred fiber type, lineage-specific gene expression in fast/slow versus fast myoblast derived primary myotubes. Sequence analysis and promoter activity assays determined that this segment contains two functional cis-regulatory elements. One element is a non-canonical E-box, and electromobility shift assays demonstrated that both cis-elements interacted with the E-protein, E47. The results indicate that primary muscle fiber type specific expression of the slow MyHC2 gene is controlled by a novel mechanism involving a transcriptional complex that includes E47 at a non-canonical E-box. PMID:26707643

  19. Association of a single nucleotide polymorphism in the 5' upstream region of the porcine myosin heavy chain 4 gene with meat quality traits in pigs.

    PubMed

    Cho, Eun-Seok; Lee, Kyung-Tai; Kim, Jun-Mo; Lee, Si-Woo; Jeon, Hyeon-Jeong; Lee, Seung-Hwan; Hong, Ki-Chang; Kim, Tae-Hun

    2016-03-01

    We identified a potential molecular marker associated with meat quality traits in the myosin heavy chain 4, MYH4 gene of Landrace pigs. Sequencing revealed a single nucleotide polymorphism (SNP; g.-1398G>T) in the 5' upstream region of MYH4. It was significantly associated with the number of type IIa muscle fibers and water-holding capacity based on filter-paper fluid uptake. The GG genotype groups had a greater number of type IIa fibers and a larger area composed of type IIa fibers than the other genotype group (P = 0.004 and P = 0.061, respectively). Expression level of MYH4 gene in the genotype TT or GT was higher than in genotype of GG (P < 0.0001). The T allele may enhance expression level of MYH4 gene and then the portion of IIb type fiber in the muscle be increased by the T allelle. Therefore, we suggest that the g.-1398G>T in the 5' upstream region of the porcine MYH4 may be used as a molecular marker for meat quality traits, although its functional effect is not defined yet. PMID:26271027

  20. Single-stranded DNA-binding proteins PURalpha and PURbeta bind to a purine-rich negative regulatory element of the alpha-myosin heavy chain gene and control transcriptional and translational regulation of the gene expression. Implications in the repression of alpha-myosin heavy chain during heart failure.

    PubMed

    Gupta, Madhu; Sueblinvong, Viranuj; Raman, Jai; Jeevanandam, Valluvan; Gupta, Mahesh P

    2003-11-01

    The alpha-myosin heavy chain is a principal molecule of the thick filament of the sarcomere, expressed primarily in cardiac myocytes. The mechanism for its cardiac-restricted expression is not yet fully understood. We previously identified a purine-rich negative regulatory (PNR) element in the first intron of the gene, which is essential for its cardiac-specific expression (Gupta, M., Zak, R., Libermann, T. A., and Gupta, M. P. (1998) Mol. Cell. Biol. 18, 7243-7258). In this study we cloned and characterized muscle and non-muscle factors that bind to this element. We show that two single-stranded DNA-binding proteins of the PUR family, PURalpha and PURbeta, which are derived from cardiac myocytes, bind to the plus strand of the PNR element. In functional assays, PURalpha and PURbeta repressed alpha-myosin heavy chain (alpha-MHC) gene expression in the presence of upstream regulatory sequences of the gene. However, from HeLa cells an Ets family of protein, Ets-related protein (ERP), binds to double-stranded PNR element. The ERP.PNR complex inhibited the activity of the basal transcription complex from homologous as well as heterologous promoters in a PNR position-independent manner, suggesting that ERP acts as a silencer of alpha-MHC gene expression in non-muscle cells. We also show that PUR proteins are capable of binding to alpha-MHC mRNA and attenuate its translational efficiency. Furthermore, we show robust expression of PUR proteins in failing hearts where alpha-MHC mRNA levels are suppressed. Together, these results reveal that (i) PUR proteins participate in transcriptional as well as translational regulation of alpha-MHC expression in cardiac myocytes and (ii) ERP may be involved in cardiac-restricted expression of the alpha-MHC gene by preventing its expression in non-muscle cells. PMID:12933792

  1. Association Analysis of Myosin Heavy-chain Genes mRNA Transcription with the Corresponding Proteins Expression of Longissimus Muscle in Growing Pigs

    PubMed Central

    Men, X. M.; Deng, B.; Tao, X.; Qi, K. K.; Xu, Z. W.

    2016-01-01

    The goal of this work was to investigate the correlations between MyHC mRNA transcription and their corresponding protein expressions in porcine longissimus muscle (LM) during postnatal growth of pigs. Five DLY (Duroc×Landrace×Yorkshire) crossbred pigs were selected, slaughtered and sampled at postnatal 7, 30, 60, 120, and 180 days, respectively. Each muscle was subjected to quantity MyHCs protein contents through an indirect enzyme-linked immunosorbent assay (ELISA), to quantity myosin heavy-chains (MyHCs) mRNA abundances using real-time polymerase chain reaction. We calculated the proportion (%) of each MyHC to total of four MyHC for two levels, respectively. Moreover, the activities of several key energy metabolism enzymes were determined in LM. The result showed that mRNA transcription and protein expression of MyHC I, IIa, IIx and IIb in LM all presented some obvious changes with postnatal aging of pigs, especially at the early stage after birth, and their mRNA transcriptions were easy to be influenced than their protein expressions. The relative proportion of each MyHC mRNA was significantly positively related to that of its corresponding protein (p<0.01), and MyHC I mRNA proportion was positively correlated with creatine kinase (CK), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) activities (p<0.05). These data suggested that MyHC mRNA transcription can be used to reflect MyHC expression, metabolism property and adaptive plasticity of porcine skeletal muscles, and MyHC mRNA composition could be a molecular index reflecting muscle fiber type characteristics. PMID:26949945

  2. Association Analysis of Myosin Heavy-chain Genes mRNA Transcription with the Corresponding Proteins Expression of Longissimus Muscle in Growing Pigs.

    PubMed

    Men, X M; Deng, B; Tao, X; Qi, K K; Xu, Z W

    2016-04-01

    The goal of this work was to investigate the correlations between MyHC mRNA transcription and their corresponding protein expressions in porcine longissimus muscle (LM) during postnatal growth of pigs. Five DLY (Duroc×Landrace×Yorkshire) crossbred pigs were selected, slaughtered and sampled at postnatal 7, 30, 60, 120, and 180 days, respectively. Each muscle was subjected to quantity MyHCs protein contents through an indirect enzyme-linked immunosorbent assay (ELISA), to quantity myosin heavy-chains (MyHCs) mRNA abundances using real-time polymerase chain reaction. We calculated the proportion (%) of each MyHC to total of four MyHC for two levels, respectively. Moreover, the activities of several key energy metabolism enzymes were determined in LM. The result showed that mRNA transcription and protein expression of MyHC I, IIa, IIx and IIb in LM all presented some obvious changes with postnatal aging of pigs, especially at the early stage after birth, and their mRNA transcriptions were easy to be influenced than their protein expressions. The relative proportion of each MyHC mRNA was significantly positively related to that of its corresponding protein (p<0.01), and MyHC I mRNA proportion was positively correlated with creatine kinase (CK), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) activities (p<0.05). These data suggested that MyHC mRNA transcription can be used to reflect MyHC expression, metabolism property and adaptive plasticity of porcine skeletal muscles, and MyHC mRNA composition could be a molecular index reflecting muscle fiber type characteristics. PMID:26949945

  3. Calcineurin-NFAT Signaling and Neurotrophins Control Transformation of Myosin Heavy Chain Isoforms in Rat Soleus Muscle in Response to Aerobic Treadmill Training

    PubMed Central

    Liu, Wenfeng; Chen, Gan; Li, Fanling; Tang, Changfa; Yin, Dazhong

    2014-01-01

    This study elucidated the role of CaN-NFAT signaling and neurotrophins on the transformation of myosin heavy chain isoforms in the rat soleus muscle fiber following aerobic exercise training. To do so, we examined the content and distribution of myosin heavy chain (MyHC) isoforms in the rat soleus muscle fiber, the activity of CaN and expression of NFATc1 in these fibers, and changes in the expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neutrophin-3 (NT-3) in the soleus and striatum following high-and medium-intensity aerobic treadmill training. Specific pathogen-free 2 month old male Sprague-Dawley (SD) rats were randomly divided into three groups: Control group (Con, n = 8), moderate-intensity aerobic exercise group (M-Ex, n = 8) and high-intensity aerobic exercise group (H-Ex, n = 8). We used ATPase staining to identify the muscle fiber type I and II, SDS-PAGE to separate and analyze the isoforms MyHCI, MyHCIIA, MyHCIIB and MyHCIIx, and performed western blots to determine the expression of NFATc1, NGF, BDNF and NT-3. CaN activity was measured using a colorimetric assay. In the soleus muscle, 8 weeks of moderate-intensity exercise can induce transformation of MyHC IIA and MyHC IIB to MyHC IIX and MyHC I (p < 0.01), while high-intensity treadmill exercise can induce transform MyHC IIx to MyHC IIB, MyHC IIA and MyHC I (p < 0.01). In comparison to the control group, CaN activity and NFATcl protein level were significantly increased in both the M-Ex and H-Ex groups (p < 0.05, p < 0.01), with a more pronounced upregulation in the M-Ex group (p < 0.05). Eight weeks of moderate- and high-intensity aerobic exercise induced the expression of NGF, BDNF and NT-3 in the soleus muscle and the striatum (p < 0.01), with the most significant increase in the H-Ex group (p < 0.01). In the rat soleus muscle, (1) CaN–NFATcl signaling contributes to the conversion of MyHC I isoform in response to moderate-intensity exercise; (2) Neurotrophins

  4. Expression of the Myosin Heavy Chain IIB Gene in Porcine Skeletal Muscle: The Role of the CArG-Box Promoter Response Element

    PubMed Central

    Brown, David M.; Brameld, John M.; Parr, Tim

    2014-01-01

    Due to its similarity to humans, the pig is increasingly being considered as a good animal model for studying a range of human diseases. Despite their physiological similarities, differential expression of the myosin heavy chain (MyHC) IIB gene (MYH4) exists in the skeletal muscles of these species, which is associated with a different muscle phenotype. The expression of different MyHC isoforms is a critical determinant of the contractile and metabolic characteristics of the muscle fibre. We aimed to elucidate whether a genomic mechanism was responsible for the drastically different expression of MYH4 between pigs and humans, thus improving our understanding of the pig as a model for human skeletal muscle research. We utilized approximately 1 kb of the MYH4 promoter from a domestic pig and a human (which do and do not express MYH4, respectively) to elucidate the role of the promoter sequence in regulating the high expression of MYH4 in porcine skeletal muscle. We identified a 3 bp genomic difference within the proximal CArG and E-box region of the MYH4 promoter of pigs and humans that dictates the differential activity of these promoters during myogenesis. Subtle species-specific genomic differences within the CArG-box region caused differential protein-DNA interactions at this site and is likely accountable for the differential MYH4 promoter activity between pigs and humans. We propose that the genomic differences identified herein explain the differential activity of the MYH4 promoter of pigs and humans, which may contribute to the differential expression patterns displayed in these otherwise physiologically similar mammals. Further, we report that both the pig and human MYH4 promoters can be induced by MyoD over-expression, but the capacity to activate the MYH4 promoter is largely influenced by the 3 bp difference located within the CArG-box region of the proximal MYH4 promoter. PMID:25469802

  5. Nonmuscle Myosin Heavy Chain IIA Is a Critical Factor Contributing to the Efficiency of Early Infection of Severe Fever with Thrombocytopenia Syndrome Virus

    PubMed Central

    Qi, Yonghe; Liu, Chenxuan; Gao, Wenqing; Chen, Pan; Fu, Liran; Peng, Bo; Wang, Haimin; Jing, Zhiyi; Zhong, Guocai

    2014-01-01

    Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel phlebovirus in the Bunyaviridae family. Most patients infected by SFTSV present with fever and thrombocytopenia, and up to 30% die due to multiple-organ dysfunction. The mechanisms by which SFTSV enters multiple cell types are unknown. SFTSV contains two species of envelope glycoproteins, Gn (44.2 kDa) and Gc (56 kDa), both of which are encoded by the M segment and are cleaved from a precursor polypeptide (about 116 kDa) in the endoplasmic reticulum (ER). Gn fused with an immunoglobulin Fc tag at its C terminus (Gn-Fc) bound to multiple cells susceptible to the infection of SFTSV and blocked viral infection of human umbilical vein endothelial cells (HUVECs). Immunoprecipitation assays following mass spectrometry analysis showed that Gn binds to nonmuscle myosin heavy chain IIA (NMMHC-IIA), a cellular protein with surface expression in multiple cell types. Small interfering RNA (siRNA) knockdown of NMMHC-IIA, but not the closely related NMMHC-IIB or NMMHC-IIC, reduced SFTSV infection, and NMMHC-IIA specific antibody blocked infection by SFTSV but not other control viruses. Overexpression of NMMHC-IIA in HeLa cells, which show limited susceptivity to SFTSV, markedly enhanced SFTSV infection of the cells. These results show that NMMHC-IIA is critical for the cellular entry of SFTSV. As NMMHC-IIA is essential for the normal functions of platelets and human vascular endothelial cells, it is conceivable that NMMHC-IIA directly contributes to the pathogenesis of SFTSV and may be a useful target for antiviral interventions against the viral infection. PMID:24155382

  6. Multiprotein complex formation at the beta myosin heavy chain distal muscle CAT element correlates with slow muscle expression but not mechanical overload responsiveness.

    PubMed

    Vyas, D R; McCarthy, J J; Tsika, G L; Tsika, R W

    2001-01-12

    To examine the role of the beta-myosin heavy chain (betaMyHC) distal muscle CAT (MCAT) element in muscle fiber type-specific expression and mechanical overload (MOV) responsiveness, we conducted transgenic and in vitro experiments. In adult transgenic mice, mutation of the distal MCAT element led to significant reductions in chloramphenicol acetyltransferase (CAT) specific activity measured in control soleus and plantaris muscles when compared with wild type transgene beta293WT but did not abolish MOV-induced CAT specific activity. Electrophoretic mobility shift assay revealed the formation of a specific low migrating nuclear protein complex (LMC) at the betaMyHC MCAT element that was highly enriched only when using either MOV plantaris or control soleus nuclear extract. Scanning mutagenesis of the betaMyHC distal MCAT element revealed that only the nucleotides comprising the core MCAT element were essential for LMC formation. The proteins within the LMC when using either MOV plantaris or control soleus nuclear extracts were antigenically related to nominal transcription enhancer factor 1 (NTEF-1), poly(ADP-ribose) polymerase (PARP), and Max. Only in vitro translated TEF-1 protein bound to the distal MCAT element, suggesting that this multiprotein complex is tethered to the DNA via TEF-1. Protein-protein interaction assays revealed interactions between nominal TEF-1, PARP, and Max. Our studies show that for transgene beta293 the distal MCAT element is not required for MOV responsiveness but suggest that a multiprotein complex likely comprised of nominal TEF-1, PARP, and Max forms at this element to contribute to basal slow fiber expression. PMID:11010974

  7. Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A.

    PubMed

    Yang, Yidai; Ye, Qilu; Jia, Zongchao; Côté, Graham P

    2015-09-25

    The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with kcat values of 1.9, 0.6, and 0.32 min(-1), respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2'/3'-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded Kd values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μM, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg(2+) ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased kcat values for kinase and ATPase activities by 3-6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site. PMID:26260792

  8. Immunohistochemical Characterization of Slow and Fast Myosin Heavy Chain Composition of Muscle Fibres in the Styloglossus Muscle of the Human and Macaque (M. rhesus)

    PubMed Central

    Sokoloff, Alan J.; Yang, Betty; Li, Haiyan; Burkholder, Thomas J.

    2007-01-01

    Objective Muscle fibre contractile diversity is thought to be increased by the hybridization of multiple myosin heavy chain (MHC) isoforms in single muscle fibres. Reports of hybrid fibres composed of MHCI and MHCII isoforms in human, but not macaque, tongue muscles, suggest a human adaptation for increased tongue muscle contractile diversity. Here we test whether hybrid fibres composed of MHCI and MHCII are unique to human tongue muscles or are present as well in the macaque. Methods MHC composition of the macaque and human styloglossus was characterized with antibodies that allowed identification of three muscle fibre phenotypes, a slow phenotype composed of MHCI, a fast phenotype composed of MHCII and a hybrid phenotype composed of MHCI and MHCII. Results The fast phenotype constitutes 68.5% of fibres in the macaque and 43.4% of fibres in the human (P<0001). The slow phenotype constitutes 20.2% of fibres in the macaque and 39.3% of fibres in the human (P<0001). The hybrid phenotype constitutes 11.2% of fibres in the macaque and 17.3% of fibres in the human (P=0002). Macaques and humans do not differ in fiber size (cross-sectional area, diameter). However, measures of fibre size differ by phenotype such that fast > hybrid > slow (P<0.05). Conclusion These data demonstrate differences in the relative percent of muscle fibre phenotypes in the macaque and human styloglossus but also demonstrate that all three phenotypes are present in both species. These data suggest a similar range of mechanical properties in styloglossus muscle fibres of the macaque and human. PMID:17210117

  9. Comparisons of different myosin heavy chain types, AMPK, and PGC-1α gene expression in the longissimus dorsi muscles in Bama Xiang and Landrace pigs.

    PubMed

    Huang, Y N; Ao, Q W; Jiang, Q Y; Guo, Y F; Lan, G Q; Jiang, H S

    2016-01-01

    Bama Xiang and Landrace pigs are the local fatty and lean breeds, respectively, in China. We compared differences in carcass traits, meat quality traits, and myosin heavy chain (MyHC) types in the longissimus dorsi muscles between Bama Xiang and Landrace pigs. This was done in pigs of the same age, using real-time PCR, to investigate the relationship between MyHC fiber types and carcass characteristics, meat quality traits, and the key factors regulating muscle fiber type. Bama Xiang pigs exhibited smaller size and slower growth than Landrace pigs (P < 0.01). We found that the superior meat quality, especially the high intramuscular fat (IMF) content in Bama Xiang pig, was related to elevated type I oxidative muscle fiber content (P < 0.01). In contrast, Landrace pig muscle had a higher glycolytic type IIb muscle fiber content (P < 0.01). MyHC I gene expression was significantly positively correlated with backfat thickness and IMF content (P < 0.01). MyHC IIb was significantly negatively correlated with IMF content (P < 0.05), and positively correlated with carcass yield (P < 0.05). AMP-activated protein kinase and peroxisome proliferator-activated receptor-g coactivator-1a are suggested to be the two key factors regulating muscle fiber type in pigs. Our results indicate that muscle fiber composition is one of the key differences leading to the differences of meat quality between Bama Xiang and Landrace pigs. These results may provide a theoretical basis for further studies of the molecular mechanism underlying the excellent meat quality of the Bama Xiang pig. PMID:27421023

  10. Differential muscular myosin heavy chain expression of the pectoral and pelvic girdles during early growth in the king penguin (Aptenodytes patagonicus) chick.

    PubMed

    Erbrech, Aude; Robin, Jean-Patrice; Guérin, Nathalie; Groscolas, René; Gilbert, Caroline; Martrette, Jean-Marc

    2011-06-01

    Continuous growth, associated with a steady parental food supply, is a general pattern in offspring development. So that young chicks can acquire their locomotor independence, this period is usually marked by a fast maturation of muscles, during which different myosin heavy chain (MyHC) isoforms are expressed. However, parental food provisioning may fluctuate seasonally, and offspring therefore face a challenge to ensure the necessary maturation of their tissues when energy is limited. To address this trade-off we investigated muscle maturation in both the pectoral and pelvic girdles of king penguin chicks. This species has an exceptionally long rearing period (1 year), which is prolonged when parental food provisioning is drastically reduced during the sub-Antarctic winter. Approximately 1 month post hatching, chicks acquire a functional pedestrian locomotion, which uses pelvic muscles, whereas swimming, which uses the pectoral muscles, only occurs 1 year later. We therefore tested the hypothesis that the MyHC content of the leg muscles reaches a mature state before those of the pectoral muscles. We found that leg muscle MyHC composition changed with the progressive acquisition of pedestrian locomotion, whereas pectoral muscle fibres reached their mature MyHC profile as early as hatching. Contrary to our predictions, the acquisition of the adult profile in pectoral muscles could be related to an early maturation of the contractile muscular proteins, presumably associated with early thermoregulatory capacities of chicks, necessary for survival in their cold environment. This differential maturation appears to reconcile both the locomotor and environmental constraints of king penguin chicks during growth. PMID:21562169

  11. Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle.

    PubMed

    Pandorf, Clay E; Jiang, Weihua; Qin, Anqi X; Bodell, Paul W; Baldwin, Kenneth M; Haddad, Fadia

    2012-04-01

    Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development. PMID:22262309

  12. Effects of pseudo-phosphorylated rat cardiac troponin T are differently modulated by α- and β-myosin heavy chain isoforms.

    PubMed

    Michael, John Jeshurun; Gollapudi, Sampath K; Chandra, Murali

    2014-01-01

    Interplay between the protein kinase C (PKC)-mediated phosphorylation of troponin T (TnT)- and myosin heavy chain (MHC)-mediated effects on thin filaments takes on a new significance because: (1) there is significant interaction between the TnT- and MHC-mediated effects on cardiac thin filaments; (2) although the phosphorylation of TnT by PKC isoforms is common to both human and rodent hearts, human hearts predominantly express β-MHC while rodent hearts predominantly express α-MHC. Therefore, we tested how α- and β-MHC isoforms differently affected the functional effects of phosphorylated TnT. Contractile measurements were made on cardiac muscle fibers from normal rats (α-MHC) and propylthiouracil-treated rats (β-MHC), reconstituted with the recombinant phosphomimetic-TnT (T204E; threonine 204 replaced by glutamate). Ca2+ -activated maximal tension decreased differently in α-MHC + T204E (~68%) and β-MHC + T204E (~35%). However, myofilament Ca2+ sensitivity decreased similarly in α-MHC + T204E and β-MHC + T204E, demonstrating that a decrease in Ca2+ sensitivity alone cannot explain the greater attenuation of tension in α-MHC + T204E. Interestingly, dynamic contractile parameters (rates of tension redevelopment, crossbridge (XB) recruitment dynamics, XB distortion dynamics, and XB detachment kinetics) decreased only in α-MHC + T204E. Thus, the transition of thin filaments from the blocked- to closed-state was attenuated in α-MHC + T204E and β-MHC + T204E, but the closed- to open-state transition was attenuated only in α-MHC + T204E. Our study demonstrates that the effects of phosphorylated TnT and MHC isoforms interact to bring about different functional states of cardiac thin filaments. PMID:25301196

  13. The functional effect of dilated cardiomyopathy mutation (R144W) in mouse cardiac troponin T is differently affected by α- and β-myosin heavy chain isoforms

    PubMed Central

    Gollapudi, Sampath K.; Tardiff, Jil C.

    2015-01-01

    Given the differential impact of α- and β-myosin heavy chain (MHC) isoforms on how troponin T (TnT) modulates contractile dynamics, we hypothesized that the effects of dilated cardiomyopathy (DCM) mutations in TnT would be altered differently by α- and β-MHC. We characterized dynamic contractile features of normal (α-MHC) and transgenic (β-MHC) mouse cardiac muscle fibers reconstituted with a mouse TnT analog (TnTR144W) of the human DCM R141W mutation. TnTR144W did not alter maximal tension but attenuated myofilament Ca2+ sensitivity (pCa50) to a similar extent in α- and β-MHC fibers. TnTR144W attenuated the speed of cross-bridge (XB) distortion dynamics (c) by 24% and the speed of XB recruitment dynamics (b) by 17% in α-MHC fibers; however, both b and c remained unaltered in β-MHC fibers. Likewise, TnTR144W attenuated the rates of XB detachment (g) and tension redevelopment (ktr) only in α-MHC fibers. TnTR144W also decreased the impact of strained XBs on the recruitment of new XBs (γ) by 30% only in α-MHC fibers. Because c, b, g, ktr, and γ are strongly influenced by thin filament-based cooperative mechanisms, we conclude that the TnTR144W- and β-MHC-mediated changes in the thin filament interact to produce a less severe functional phenotype, compared with that brought about by TnTR144W and α-MHC. These observations provide a basis for lower mortality rates of humans (β-MHC) harboring the TnTR141W mutant compared with transgenic mouse studies. Our findings strongly suggest that some caution is necessary when extrapolating data from transgenic mouse studies to human hearts. PMID:25681424

  14. Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by β2-adrenoceptor stimulation.

    PubMed

    Ohnuki, Yoshiki; Umeki, Daisuke; Mototani, Yasumasa; Jin, Huiling; Cai, Wenqian; Shiozawa, Kouichi; Suita, Kenji; Saeki, Yasutake; Fujita, Takayuki; Ishikawa, Yoshihiro; Okumura, Satoshi

    2014-12-15

    The predominant isoform of β-adrenoceptor (β-AR) in skeletal muscle is β2-AR and that in the cardiac muscle is β1-AR. We have reported that Epac1 (exchange protein directly activated by cAMP 1), a new protein kinase A-independent cAMP sensor, does not affect cardiac hypertrophy in response to pressure overload or chronic isoproterenol (isoprenaline) infusion. However, the role of Epac1 in skeletal muscle hypertrophy remains poorly understood. We thus examined the effect of disruption of Epac1, the major Epac isoform in skeletal muscle, on masseter muscle hypertrophy induced by chronic β2-AR stimulation with clenbuterol (CB) in Epac1-null mice (Epac1KO). The masseter muscle weight/tibial length ratio was similar in wild-type (WT) and Epac1KO at baseline and was significantly increased in WT after CB infusion, but this increase was suppressed in Epac1KO. CB treatment significantly increased the proportion of myosin heavy chain (MHC) IIb at the expense of that of MHC IId/x in both WT and Epac1KO, indicating that Epac1 did not mediate the CB-induced MHC isoform transition towards the faster isoform. The mechanism of suppression of CB-mediated hypertrophy in Epac1KO is considered to involve decreased activation of Akt signalling. In addition, CB-induced histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy, was suppressed in Epac1KO. Our findings suggest that Epac1 plays a role in β2-AR-mediated masseter muscle hypertrophy, probably through activation of both Akt signalling and CaMKII/HDAC4 signalling. PMID:25344550

  15. Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

    SciTech Connect

    Eom, Young Woo; Lee, Jong Eun; Yang, Mal Sook; Jang, In Keun; Kim, Hyo Eun; Lee, Doo Hoon; Kim, Young Jin; Park, Won Jin; Kong, Jee Hyun; Shim, Kwang Yong; Lee, Jong In; Kim, Hyun Soo

    2011-04-29

    Highlights: {yields} hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. {yields} Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. {yields} hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

  16. Myosin‑II heavy chain and formin mediate the targeting of myosin essential light chain to the division site before and during cytokinesis.

    PubMed

    Feng, Zhonghui; Okada, Satoshi; Cai, Guoping; Zhou, Bing; Bi, Erfei

    2015-04-01

    MLC1 is a haploinsufficient gene encoding the essential light chain for Myo1, the sole myosin‑II heavy chain in the budding yeast Saccharomyces cerevisiae. Mlc1 defines an essential hub that coordinates actomyosin ring function, membrane trafficking, and septum formation during cytokinesis by binding to IQGAP, myosin‑II, and myosin‑V. However, the mechanism of how Mlc1 is targeted to the division site during the cell cycle remains unsolved. By constructing a GFP‑tagged MLC1 under its own promoter control and using quantitative live‑cell imaging coupled with yeast mutants, we found that septin ring and actin filaments mediate the targeting of Mlc1 to the division site before and during cytokinesis, respectively. Both mechanisms contribute to and are collectively required for the accumulation of Mlc1 at the division site during cytokinesis. We also found that Myo1 plays a major role in the septin‑dependent Mlc1 localization before cytokinesis, whereas the formin Bni1 plays a major role in the actin filament-dependent Mlc1 localization during cytokinesis. Such a two‑tiered mechanism for Mlc1 localization is presumably required for the ordered assembly and robustness of cytokinesis machinery and is likely conserved across species. PMID:25631819

  17. Differential effects of short-term β agonist and growth hormone treatments on expression of myosin heavy chain IIB and associated metabolic genes in sheep muscle.

    PubMed

    Hemmings, K M; Daniel, Z C T R; Buttery, P J; Parr, T; Brameld, J M

    2015-02-01

    Growth hormone (GH) and β agonists increase muscle mass, but the mechanisms for this response are unclear and the magnitude of response is thought to vary with age of animal. To investigate the mechanisms driving the muscle response to these agents, we examined the effects of short-term (6 day) administration of GH or cimaterol (a β2-adrenergic agonist, BA) on skeletal muscle phenotype in both young (day 60) and mature (day 120) lambs. Expression of myosin heavy chain (MyHC) isoforms were measured in Longissimus dorsi (LD), Semitendinosus (ST) and Supraspinatus (SS) muscles as markers of fibre type and metabolic enzyme activities were measured in LD. To investigate potential mechanisms regulating the changes in fibre type/metabolism, expression or activity of a number of signalling molecules were examined in LD. There were no effects of GH administration on MyHC isoform expression at either the mRNA or protein level in any of the muscles. However, BA treatment induced a proportional change in MyHC mRNA expression at both ages, with the %MyHCI and/or IIA mRNA being significantly decreased in all three muscles and %MyHCIIX/IIB mRNA significantly increased in the LD and ST. BA treatment induced de novo expression of MyHCIIB mRNA in LD, the fastest isoform not normally expressed in sheep LD, as well as increasing expression in the other two muscles. In the LD, the increased expression of the fastest MyHC isoforms (IIX and IIB) was associated with a decrease in isocitrate dehydrogenase activity, but no change in lactate dehydrogenase activity, indicating a reduced capacity for oxidative metabolism. In both young and mature lambs, changes in expression of metabolic regulatory factors were observed that might induce these changes in muscle metabolism/fibre type. In particular, BA treatment decreased PPAR-γ coactivator-1β mRNA and increased receptor-interacting protein 140 mRNA. The results suggest that the two agents work via different mechanisms or over different

  18. Immunogenicity and Protective Efficacy of Brugia malayi Heavy Chain Myosin as Homologous DNA, Protein and Heterologous DNA/Protein Prime Boost Vaccine in Rodent Model.

    PubMed

    Gupta, Jyoti; Pathak, Manisha; Misra, Sweta; Misra-Bhattacharya, Shailja

    2015-01-01

    We earlier demonstrated the immunoprophylactic efficacy of recombinant heavy chain myosin (Bm-Myo) of Brugia malayi (B. malayi) in rodent models. In the current study, further attempts have been made to improve this efficacy by employing alternate approaches such as homologous DNA (pcD-Myo) and heterologous DNA/protein prime boost (pcD-Myo+Bm-Myo) in BALB/c mouse model. The gene bm-myo was cloned in a mammalian expression vector pcDNA 3.1(+) and protein expression was confirmed in mammalian Vero cell line. A significant degree of protection (79.2%±2.32) against L3 challenge in pcD-Myo+Bm-Myo immunized group was observed which was much higher than that exerted by Bm-Myo (66.6%±2.23) and pcD-Myo (41.6%±2.45). In the heterologous immunized group, the percentage of peritoneal leukocytes such as macrophages, neutrophils, B cells and T cells marginally increased and their population augmented further significantly following L3 challenge. pcD-Myo+Bm-Myo immunization elicited robust cellular and humoral immune responses as compared to pcD-Myo and Bm-Myo groups as evidenced by an increased accumulation of CD4+, CD8+ T cells and CD19+ B cells in the mouse spleen and activation of peritoneal macrophages. Though immunized animals produced antigen-specific IgG antibodies and isotypes, sera of mice receiving pcD-Myo+Bm-Myo or Bm-Myo developed much higher antibody levels than other groups and there was profound antibody-dependent cellular adhesion and cytotoxicity (ADCC) to B. malayi infective larvae (L3). pcD-Myo+Bm-Myo as well as Bm-Myo mice generated a mixed T helper cell phenotype as evidenced by the production of both pro-inflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines. Mice receiving pcD-Myo on contrary displayed a polarized pro-inflammatory immune response. The findings suggest that the priming of animals with DNA followed by protein booster generates heightened and mixed pro- and anti-inflammatory immune responses that are capable of providing

  19. Immunogenicity and Protective Efficacy of Brugia malayi Heavy Chain Myosin as Homologous DNA, Protein and Heterologous DNA/Protein Prime Boost Vaccine in Rodent Model

    PubMed Central

    Gupta, Jyoti; Pathak, Manisha; Misra, Sweta; Misra-Bhattacharya, Shailja

    2015-01-01

    We earlier demonstrated the immunoprophylactic efficacy of recombinant heavy chain myosin (Bm-Myo) of Brugia malayi (B. malayi) in rodent models. In the current study, further attempts have been made to improve this efficacy by employing alternate approaches such as homologous DNA (pcD-Myo) and heterologous DNA/protein prime boost (pcD-Myo+Bm-Myo) in BALB/c mouse model. The gene bm-myo was cloned in a mammalian expression vector pcDNA 3.1(+) and protein expression was confirmed in mammalian Vero cell line. A significant degree of protection (79.2%±2.32) against L3 challenge in pcD-Myo+Bm-Myo immunized group was observed which was much higher than that exerted by Bm-Myo (66.6%±2.23) and pcD-Myo (41.6%±2.45). In the heterologous immunized group, the percentage of peritoneal leukocytes such as macrophages, neutrophils, B cells and T cells marginally increased and their population augmented further significantly following L3 challenge. pcD-Myo+Bm-Myo immunization elicited robust cellular and humoral immune responses as compared to pcD-Myo and Bm-Myo groups as evidenced by an increased accumulation of CD4+, CD8+ T cells and CD19+ B cells in the mouse spleen and activation of peritoneal macrophages. Though immunized animals produced antigen-specific IgG antibodies and isotypes, sera of mice receiving pcD-Myo+Bm-Myo or Bm-Myo developed much higher antibody levels than other groups and there was profound antibody-dependent cellular adhesion and cytotoxicity (ADCC) to B. malayi infective larvae (L3). pcD-Myo+Bm-Myo as well as Bm-Myo mice generated a mixed T helper cell phenotype as evidenced by the production of both pro-inflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines. Mice receiving pcD-Myo on contrary displayed a polarized pro-inflammatory immune response. The findings suggest that the priming of animals with DNA followed by protein booster generates heightened and mixed pro- and anti-inflammatory immune responses that are capable of providing

  20. Increase in cardiac myosin heavy-chain (MyHC) alpha protein isoform in hibernating ground squirrels, with echocardiographic visualization of ventricular wall hypertrophy and prolonged contraction

    PubMed Central

    Nelson, O. Lynne; Rourke, Bryan C.

    2013-01-01

    myosin heavy-chain (MyHC) isoforms in a separate cohort of squirrels over 5 months, including time points before hibernation, during hibernation and just prior to emergence. Hibernating individuals were maintained in both a 4°C cold room and a 20°C warm room. Measured by SDS-PAGE, relative percentages of cardiac MyHC alpha were increased during hibernation, at both hibernacula temperatures. A potential increase in contractile speed, and power, from more abundant MyHC alpha may aid force generation at low temperature and at low heart rates. Unlike many models of cardiomyopathies where the alpha isoform is replaced by the beta isoform in order to reduce oxygen consumption, ground squirrels demonstrate a potential cardioprotective mechanism to maintain cardiac output during torpor. PMID:24072796

  1. Rat cardiac troponin T mutation (F72L)-mediated impact on thin filament cooperativity is divergently modulated by α- and β-myosin heavy chain isoforms.

    PubMed

    Chandra, Vikram; Gollapudi, Sampath K; Chandra, Murali

    2015-10-01

    The primary causal link between disparate effects of human hypertrophic cardiomyopathy (HCM)-related mutations in troponin T (TnT) and α- and β-myosin heavy chain (MHC) isoforms on cardiac contractile phenotype remains poorly understood. Given the divergent impact of α- and β-MHC on the NH2-terminal extension (44-73 residues) of TnT, we tested if the effects of the HCM-linked mutation (TnTF70L) were differentially altered by α- and β-MHC. We hypothesized that the emergence of divergent thin filament cooperativity would lead to contrasting effects of TnTF70L on contractile function in the presence of α- and β-MHC. The rat TnT analog of the human F70L mutation (TnTF72L) or the wild-type rat TnT (TnTWT) was reconstituted into demembranated muscle fibers from normal (α-MHC) and propylthiouracil-treated (β-MHC) rat hearts to measure steady-state and dynamic contractile function. TnTF72L-mediated effects on tension, myofilament Ca(2+) sensitivity, myofilament cooperativity, rate constants of cross-bridge (XB) recruitment dynamics, and force redevelopment were divergently modulated by α- and β-MHC. TnTF72L increased the rate of XB distortion dynamics by 49% in α-MHC fibers but had no effect in β-MHC fibers; these observations suggest that TnTF72L augmented XB detachment kinetics in α-MHC, but not β-MHC, fibers. TnTF72L increased the negative impact of strained XBs on the force-bearing XBs by 39% in α-MHC fibers but had no effect in β-MHC fibers. Therefore, TnTF72L leads to contractile changes that are linked to dilated cardiomyopathy in the presence of α-MHC. On the other hand, TnTF72L leads to contractile changes that are linked to HCM in the presence of β-MHC. PMID:26342069

  2. The heavy chain has its day

    PubMed Central

    Dulyaninova, Natalya G; Bresnick, Anne R

    2013-01-01

    Nonmuscle myosin-II is an actin-based motor that converts chemical energy into force and movement, and thus functions as a key regulator of the eukaryotic cytoskeleton. Although it is established that phosphorylation on the regulatory light chain increases the actin-activated MgATPase activity of the motor and promotes myosin-II filament assembly, studies have begun to characterize alternative mechanisms that regulate filament assembly and disassembly. These investigations have revealed that all three nonmuscle myosin-II isoforms are subject to additional regulatory controls, which impact diverse cellular processes. In this review, we discuss current knowledge on mechanisms that regulate the oligomerization state of nonmuscle myosin-II filaments by targeting the myosin heavy chain. PMID:24002531

  3. Skeletal muscle myosin light chains are essential for physiological speeds of shortening.

    PubMed

    Lowey, S; Waller, G S; Trybus, K M

    1993-09-30

    In muscle each myosin head contains a regulatory light chain (LC2) that is wrapped around the head/rod junction, and an alkali light chain that is distal to LC2 (ref. 1). The role of these light chains in vertebrate skeletal muscle myosin has remained obscure. Here we prepare heavy chains that are free of both light chains in order to determine by a motility assay whether the light chains are necessary for movement. We find that removal of light chains from myosin reduces the velocity of actin filaments from 8.8 microns s-1 to 0.8 microns s-1 without significantly decreasing the ATPase activity. Reconstitution of myosin with LC2 or alkali light chain increases filament velocity to intermediate rates, and readdition of both classes of light chains fully restores the original sliding velocity. We conclude that even though the light chains are not essential for enzymatic activity, light-chain/heavy-chain interactions play an important part in the conversion of chemical energy into movement. PMID:8413589

  4. Overexpression of smooth muscle myosin heavy chain leads to activation of the unfolded protein response and autophagic turnover of thick filament-associated proteins in vascular smooth muscle cells.

    PubMed

    Kwartler, Callie S; Chen, Jiyuan; Thakur, Dhananjay; Li, Shumin; Baskin, Kedryn; Wang, Shanzhi; Wang, Zhao V; Walker, Lori; Hill, Joseph A; Epstein, Henry F; Taegtmeyer, Heinrich; Milewicz, Dianna M

    2014-05-16

    Duplications spanning nine genes at the genomic locus 16p13.1 predispose individuals to acute aortic dissections. The most likely candidate gene in this region leading to the predisposition for dissection is MYH11, which encodes smooth muscle myosin heavy chain (SM-MHC). The effects of increased expression of MYH11 on smooth muscle cell (SMC) phenotypes were explored using mouse aortic SMCs with transgenic overexpression of one isoform of SM-MHC. We found that these cells show increased expression of Myh11 and myosin filament-associated contractile genes at the message level when compared with control SMCs, but not at the protein level due to increased protein degradation. Increased expression of Myh11 resulted in endoplasmic reticulum (ER) stress in SMCs, which led to a paradoxical decrease of protein levels through increased autophagic degradation. An additional consequence of ER stress in SMCs was increased intracellular calcium ion concentration, resulting in increased contractile signaling and contraction. The increased signals for contraction further promote transcription of contractile genes, leading to a feedback loop of metabolic abnormalities in these SMCs. We suggest that overexpression of MYH11 can lead to increased ER stress and autophagy, findings that may be globally implicated in disease processes associated with genomic duplications. PMID:24711452

  5. Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load.

    PubMed

    Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R

    2015-08-15

    Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin's ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an α-actinin frictional load. Porcine cardiac β-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31-41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level. PMID:26116789

  6. Point Mutations in Human β Cardiac Myosin Heavy Chain Have Differential Effects on Sarcomeric Structure and Assembly: An ATP Binding Site Change Disrupts Both Thick and Thin Filaments, Whereas Hypertrophic Cardiomyopathy Mutations Display Normal Assembly

    PubMed Central

    Becker, K. David; Gottshall, Kim R.; Hickey, Reed; Perriard, Jean-Claude; Chien, Kenneth R.

    1997-01-01

    Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including β cardiac myosin heavy chain (βMHC). To determine whether point mutations in human βMHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human βMHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human βMHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse βMHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human βMHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type βMHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human βMHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a

  7. Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load

    PubMed Central

    Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R.

    2016-01-01

    Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin’s ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an α-actinin frictional load. Porcine cardiac β-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31–41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level. PMID:26116789

  8. Tyrosine phosphorylation/dephosphorylation of myosin II essential light chains of Entamoeba histolytica trophozoites regulates their motility.

    PubMed

    Bonilla-Moreno, Raúl; Pérez-Yépez, Eloy-Andrés; Villegas-Sepúlveda, Nicolás; Morales, Fernando O; Meza, Isaura

    2016-08-01

    Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites. PMID:27318258

  9. Ultraviolet-induced vanadate-dependent modification and cleavage of skeletal myosin subfragment 1 heavy chain. 2. Oxidation of serine in the 23-kDa NH/sub 2/-terminal tryptic peptide

    SciTech Connect

    Cremo, C.R.; Grammer, J.C.; Yount, R.G.

    1988-11-01

    Myosin subfragment 1 (S1) can be specifically photomodified at the active site without polypeptide chain cleavage by irradiating the stable MgADP-orthovanadate-S1 complex with UV light above 300 nm. Here, the UV spectral properties of photomodified S1 were used to determined the nature and location of the photomodified residue(s) within S1. By comparison of the unusual pH dependence of the UV absorption spectrum of the photomodified S1 to that of the S1-MgADP-V/sub i/ complex as a control the photomodified residue(s) was (were) localized to the 23-kDa NH/sub 2/-terminal tryptic peptide of the heavy chain. NaBH/sub 4/ reduced the photomodified S1, but not the control, to regenerate the original spectral properties and ATPase activities of the unmodified S1. Amino acid analysis of photomodified S1 reduced with NaB/sup 3/H/sub 4/ gave only (/sup 3/H)serine, suggesting the hydroxyl group of serine had been oxidized to a serine aldehyde. The pH dependence of the absorption spectrum of the photomodified enzyme can be explained by an equilibrium between a chromophoric enolate anion of the serine aldehyde (favored in base) and less chromophoric keto and enol forms (favored in acid). The oxidized serine(s) was (were) shown to be directly involved with the vanadate-dependent photocleavage of the S1 heavy chain previously described by Grammer et al. (1988). This serine(s) is (are) likely to be important to the binding and hydrolysis of the ..gamma..-PO/sub 4/ of ATP at the active site of S1.

  10. A Novel Alpha Cardiac Actin (ACTC1) Mutation Mapping to a Domain in Close Contact with Myosin Heavy Chain Leads to a Variety of Congenital Heart Defects, Arrhythmia and Possibly Midline Defects

    PubMed Central

    Augière, Céline; Mégy, Simon; El Malti, Rajae; Boland, Anne; El Zein, Loubna; Verrier, Bernard; Mégarbané, André; Deleuze, Jean-François; Bouvagnet, Patrice

    2015-01-01

    Background A Lebanese Maronite family presented with 13 relatives affected by various congenital heart defects (mainly atrial septal defects), conduction tissue anomalies and midline defects. No mutations were found in GATA4 and NKX2-5. Methods and Results A set of 399 poly(AC) markers was used to perform a linkage analysis which peaked at a 2.98 lod score on the long arm of chromosome 15. The haplotype analysis delineated a 7.7 meganucleotides genomic interval which included the alpha-cardiac actin gene (ACTC1) among 36 other protein coding genes. A heterozygous missense mutation was found (c.251T>C, p.(Met84Thr)) in the ACTC1 gene which changed a methionine residue conserved up to yeast. This mutation was absent from 1000 genomes and exome variant server database but segregated perfectly in this family with the affection status. This mutation and 2 other ACTC1 mutations (p.(Glu101Lys) and p.(Met125Val)) which result also in congenital heart defects are located in a region in close apposition to a myosin heavy chain head region by contrast to 3 other alpha-cardiac actin mutations (p.(Ala297Ser),p.(Asp313His) and p.(Arg314His)) which result in diverse cardiomyopathies and are located in a totally different interaction surface. Conclusions Alpha-cardiac actin mutations lead to congenital heart defects, cardiomyopathies and eventually midline defects. The consequence of an ACTC1 mutation may in part be dependent on the interaction surface between actin and myosin. PMID:26061005

  11. Stretch activates myosin light chain kinase in arterial smooth muscle

    SciTech Connect

    Barany, K.; Rokolya, A.; Barany, M. )

    1990-11-30

    Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol (32P)phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the (32P)phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in (32P)phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively.

  12. Myosin Light Chain Kinase (MLCK) Regulates Cell Migration in a Myosin Regulatory Light Chain Phosphorylation-independent Mechanism*

    PubMed Central

    Chen, Chen; Tao, Tao; Wen, Cheng; He, Wei-Qi; Qiao, Yan-Ning; Gao, Yun-Qian; Chen, Xin; Wang, Pei; Chen, Cai-Ping; Zhao, Wei; Chen, Hua-Qun; Ye, An-Pei; Peng, Ya-Jing; Zhu, Min-Sheng

    2014-01-01

    Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration. PMID:25122766

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

    PubMed

    Kampourakis, Thomas; Irving, Malcolm

    2015-08-01

    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. PMID:26057075

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

    PubMed Central

    Kampourakis, Thomas; Irving, Malcolm

    2015-01-01

    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. PMID:26057075

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

    PubMed Central

    Jancso, A; Szent-Györgyi, A G

    1994-01-01

    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. Images PMID:8090720

  16. Effects of myosin light chain phosphorylation on length-dependent myosin kinetics in skinned rat myocardium.

    PubMed

    Pulcastro, Hannah C; Awinda, Peter O; Breithaupt, Jason J; Tanner, Bertrand C W

    2016-07-01

    Myosin force production is Ca(2+)-regulated by thin-filament proteins and sarcomere length, which together determine the number of cross-bridge interactions throughout a heartbeat. Ventricular myosin regulatory light chain-2 (RLC) binds to the neck of myosin and modulates contraction via its phosphorylation state. Previous studies reported regional variations in RLC phosphorylation across the left ventricle wall, suggesting that RLC phosphorylation could alter myosin behavior throughout the heart. We found that RLC phosphorylation varied across the left ventricle wall and that RLC phosphorylation was greater in the right vs. left ventricle. We also assessed functional consequences of RLC phosphorylation on Ca(2+)-regulated contractility as sarcomere length varied in skinned rat papillary muscle strips. Increases in RLC phosphorylation and sarcomere length both led to increased Ca(2+)-sensitivity of the force-pCa relationship, and both slowed cross-bridge detachment rate. RLC-phosphorylation slowed cross-bridge rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 μm sarcomere length, whereas RLC phosphorylation only slowed cross-bridge MgATP binding rate (∼55%) at 2.2 μm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles. PMID:26763941

  17. Constitutive phosphorylation of cardiac myosin regulatory light chain in vivo.

    PubMed

    Chang, Audrey N; Battiprolu, Pavan K; Cowley, Patrick M; Chen, Guohua; Gerard, Robert D; Pinto, Jose R; Hill, Joseph A; Baker, Anthony J; Kamm, Kristine E; Stull, James T

    2015-04-24

    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 Ca(2+) 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. PMID:25733667

  18. Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions.

    PubMed

    Duggal, D; Nagwekar, J; Rich, R; Huang, W; Midde, K; Fudala, R; Das, H; Gryczynski, I; Szczesna-Cordary, D; Borejdo, J

    2015-05-15

    Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to ∼20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contraction-relaxation cycle may contribute to altered contractility and the development of FHC. PMID:25770245

  19. Polymorphisms in the Non-Muscle Myosin Heavy Chain Gene (MYH9) Are Associated with Lower Glomerular Filtration Rate in Mixed Ancestry Diabetic Subjects from South Africa

    PubMed Central

    Matsha, Tandi Edith; Masconi, Katya; Yako, Yandiswa Yolanda; Hassan, Mogamat Shafick; Macharia, Muiriri; Erasmus, Rajiv Timothy; Kengne, Andre Pascal

    2012-01-01

    Objective Though single nucleotide polymorphisms (SNPs) in the non-muscle myosin gene (MYH9) have been reported to explain most of the excess risk of nondiabetic chronic kidney disease (CKD), in African-Americans, some studies have also shown associations with diabetic end-stage renal disease. We investigated the association of MYH9 SNPs with renal traits in a mixed-ancestry South African population prone to diabetes. Research Design and Methods Three SNPs known to be associated with CKD (rs4821480, rs5756152 and rs12107) were genotyped using Taqman assay in 716 adults (198 with diabetes) from the Bellville-South community, Cape Town. Glomerular filtration rate was estimated (eGFR) and urinary albumin/creatinine ratio (ACR) assessed. Multivariable regressions were used to relate the SNPs with renal traits. Results Mean age was 53.6 years, with the expected differences observed in characteristics by diabetic status. Significant associations were found between rs575152 and serum creatinine, and eGFR in the total population, and in diabetic participants (all p≤0.003), but not in non-diabetics (all p≥0.16), with significant interactions by diabetes status (interaction-p≤0.009). The association with ACR was borderline in diabetic participants (p = 0.05) and non-significant in non-diabetics (p = 0.85), with significant interaction (interaction p = 0.02). rs12107 was associated with fasting-, 2-hour glucose and HbA1c in diabetic participants only (interaction-p≤0.003), but not with renal traits. Conclusion MYH9 SNPs were associated with renal traits only in diabetic participants in this population. Our findings and other studies suggest that MYH9 may have a broader genetic risk effect on kidney diseases. PMID:23285077

  20. The effect of cardiomyopathy mutation (R97L) in mouse cardiac troponin T on the muscle length-mediated recruitment of crossbridges is modified divergently by α- and β-myosin heavy chain.

    PubMed

    Gollapudi, Sampath K; Chandra, Murali

    2016-07-01

    Hypertrophic cardiomyopathy mutations in cardiac troponin T (TnT) lead to sudden cardiac death. Augmented myofilament Ca(2+) sensitivity is a common feature in TnT mutants, but such observations fail to provide a rational explanation for severe cardiac phenotypes. To better understand the mutation-induced effect on the cardiac phenotype, it is imperative to determine the effects on dynamic contractile features such as the muscle length (ML)-mediated activation against α- and β-myosin heavy chain (MHC) isoforms. α- and β-MHC are not only differentially expressed in rodent and human hearts, but they also modify ML-mediated activation differently. Mouse analog of human TnTR94L (TnTR97L) or wild-type TnT was reconstituted into de-membranated muscle fibers from normal (α-MHC) and transgenic (β-MHC) mouse hearts. TnTR97L augmented myofilament Ca(2+) sensitivity by a similar amount in α- and β-MHC fibers. However, TnTR97L augmented the negative impact of strained crossbridges on other crossbridges (γ) by 22% in α-MHC fibers, but attenuated γ by 21% in β-MHC fibers. TnTR97L decreased the magnitude of ML-mediated recruitment of crossbridges (ER) by 37% in α-MHC fibers, but increased ER by 35% in β-MHC fibers. We provide a mechanistic basis for the TnTR97L-induced effects in α- and β-MHC fibers and discuss the relevance to human hearts. PMID:26792537

  1. Increased cardiac alpha-myosin heavy chain in left atria and decreased myocardial insulin-like growth factor (Igf-I) expression accompany low heart rate in hibernating grizzly bears.

    PubMed

    Barrows, N D; Nelson, O L; Robbins, C T; Rourke, B C

    2011-01-01

    Grizzly bears (Ursus arctos horribilis) tolerate extended periods of extremely low heart rate during hibernation without developing congestive heart failure or cardiac chamber dilation. Left ventricular atrophy and decreased left ventricular compliance have been reported in this species during hibernation. We evaluated the myocardial response to significantly reduced heart rate during hibernation by measuring relative myosin heavy-chain (MyHC) isoform expression and expression of a set of genes important to muscle plasticity and mass regulation in the left atria and left ventricles of active and hibernating bears. We supplemented these data with measurements of systolic and diastolic function via echocardiography in unanesthetized grizzly bears. Atrial strain imaging revealed decreased atrial contractility, decreased expansion/reservoir function (increased atrial stiffness), and decreased passive-filling function (increased ventricular stiffness) in hibernating bears. Relative MyHC-α protein expression increased significantly in the atrium during hibernation. The left ventricle expressed 100% MyHC-β protein in both groups. Insulin-like growth factor (IGF-I) mRNA expression was reduced by ∼50% in both chambers during hibernation, consistent with the ventricular atrophy observed in these bears. Interestingly, mRNA expression of the atrophy-related ubiquitin ligases Muscle Atrophy F-box (MAFBx) and Muscle Ring Finger 1 did not increase, nor did expression of myostatin or hypoxia-inducible factor 1α (HIF-1α). We report atrium-specific decreases of 40% and 50%, respectively, in MAFBx and creatine kinase mRNA expression during hibernation. Decreased creatine kinase expression is consistent with lowered energy requirements and could relate to reduced atrial emptying function during hibernation. Taken together with our hemodynamic assessment, these data suggest a potential downregulation of atrial chamber function during hibernation to prevent fatigue and dilation

  2. N-Terminus of Cardiac Myosin Essential Light Chain Modulates Myosin Step-Size.

    PubMed

    Wang, Yihua; Ajtai, Katalin; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Burghardt, Thomas P

    2016-01-12

    Muscle myosin cyclically hydrolyzes ATP to translate actin. Ventricular cardiac myosin (βmys) moves actin with three distinct unitary step-sizes resulting from its lever-arm rotation and with step-frequencies that are modulated in a myosin regulation mechanism. The lever-arm associated essential light chain (vELC) binds actin by its 43 residue N-terminal extension. Unitary steps were proposed to involve the vELC N-terminal extension with the 8 nm step engaging the vELC/actin bond facilitating an extra ∼19 degrees of lever-arm rotation while the predominant 5 nm step forgoes vELC/actin binding. A minor 3 nm step is the unlikely conversion of the completed 5 to the 8 nm step. This hypothesis was tested using a 17 residue N-terminal truncated vELC in porcine βmys (Δ17βmys) and a 43 residue N-terminal truncated human vELC expressed in transgenic mouse heart (Δ43αmys). Step-size and step-frequency were measured using the Qdot motility assay. Both Δ17βmys and Δ43αmys had significantly increased 5 nm step-frequency and coincident loss in the 8 nm step-frequency compared to native proteins suggesting the vELC/actin interaction drives step-size preference. Step-size and step-frequency probability densities depend on the relative fraction of truncated vELC and relate linearly to pure myosin species concentrations in a mixture containing native vELC homodimer, two truncated vELCs in the modified homodimer, and one native and one truncated vELC in the heterodimer. Step-size and step-frequency, measured for native homodimer and at two or more known relative fractions of truncated vELC, are surmised for each pure species by using a new analytical method. PMID:26671638

  3. Heavy Chain Diseases

    MedlinePlus

    ... cells often prevents proper absorption of nutrients from food (malabsorption), resulting in severe diarrhea and weight loss. A rare form that affects the respiratory tract also exists. Blood tests are done when alpha heavy chain disease is suspected. Serum protein electrophoresis, measurement of ...

  4. Novel familial dilated cardiomyopathy mutation in MYL2 affects the structure and function of myosin regulatory light chain.

    PubMed

    Huang, Wenrui; Liang, Jingsheng; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Zhou, Zhiqun; Morales, Ana; McBride, Kim L; Fitzgerald-Butt, Sara M; Hershberger, Ray E; Szczesna-Cordary, Danuta

    2015-06-01

    Dilated cardiomyopathy (DCM) is a disease of the myocardium characterized by left ventricular dilatation and diminished contractile function. Here we describe a novel DCM mutation in the myosin regulatory light chain (RLC), in which aspartic acid at position 94 is replaced by alanine (D94A). The mutation was identified by exome sequencing of three adult first-degree relatives who met formal criteria for idiopathic DCM. To obtain insight into the functional significance of this pathogenic MYL2 variant, we cloned and purified the human ventricular RLC wild-type (WT) and D94A mutant proteins, and performed in vitro experiments using RLC-mutant or WT-reconstituted porcine cardiac preparations. The mutation induced a reduction in the α-helical content of the RLC, and imposed intra-molecular rearrangements. The phosphorylation of RLC by Ca²⁺/calmodulin-activated myosin light chain kinase was not affected by D94A. The mutation was seen to impair binding of RLC to the myosin heavy chain, and its incorporation into RLC-depleted porcine myosin. The actin-activated ATPase activity of mutant-reconstituted porcine cardiac myosin was significantly higher compared with ATPase of wild-type. No changes in the myofibrillar ATPase-pCa relationship were observed in wild-type- or D94A-reconstituted preparations. Measurements of contractile force showed a slightly reduced maximal tension per cross-section of muscle, with no change in the calcium sensitivity of force in D94A-reconstituted skinned porcine papillary muscle strips compared with wild-type. Our data indicate that subtle structural rearrangements in the RLC molecule, followed by its impaired interaction with the myosin heavy chain, may trigger functional abnormalities contributing to the DCM phenotype. PMID:25825243

  5. Stimulatory and inhibitory mechanisms of slow muscle-specific myosin heavy chain gene expression in fish: Transient and transgenic analysis of torafugu MYH{sub M86-2} promoter in zebrafish embryos

    SciTech Connect

    Asaduzzaman, Md.; Kinoshita, Shigeharu; Bhuiyan, Sharmin Siddique; Asakawa, Shuichi; Watabe, Shugo

    2013-04-01

    The myosin heavy chain gene, MYH{sub M86-2}, exhibited restricted expression in slow muscle fibers of torafugu embryos and larvae, suggesting its functional roles for embryonic and larval muscle development. However, the transcriptional mechanisms involved in its expression are still ambiguous. The present study is the first extensive analysis of slow muscle-specific MYH{sub M86-2} promoter in fish for identifying the cis-elements that are crucial for its expression. Combining both transient transfection and transgenic approaches, we demonstrated that the 2614 bp 5′-flanking sequences of MYH{sub M86-2} contain a sufficient promoter activity to drive gene expression specific to superficial slow muscle fibers. By cyclopamine treatment, we also demonstrated that the differentiation of such superficial slow muscle fibers depends on hedgehog signaling activity. The deletion analyses defined an upstream fragment necessary for repressing ectopic MYH{sub M86-2} expression in the fast muscle fibers. The transcriptional mechanism that prevents MYH{sub M86-2} expression in the fast muscle fibers is mediated through Sox6 binding elements. We also demonstrated that Sox6 may function as a transcriptional repressor of MYH{sub M86-2} expression. We further discovered that nuclear factor of activated T cells (NFAT) binding elements plays a key role and myocyte enhancer factor-2 (MEF2) binding elements participate in the transcriptional regulation of MYH{sub M86-2} expression. - Highlights: ► MYH{sub M86-2} is highly expressed in slow muscle fibers of torafugu embryos and larvae. ► MYH{sub M86-2} promoter activity depends on the hedgehog signaling. ► Sox6 binding elements inhibits MYH{sub M86-2} expression in fast muscle fibers. ► Sox6 elements function as transcriptional repressor of MYH{sub M86-2} promoter activity. ► NFAT and MEF2 binding elements play a key role for directing MYH{sub M86-2} expression.

  6. Potentiation in mouse lumbrical muscle without myosin light chain phosphorylation: is resting calcium responsible?

    PubMed

    Smith, Ian C; Gittings, William; Huang, Jian; McMillan, Elliott M; Quadrilatero, Joe; Tupling, A Russell; Vandenboom, Rene

    2013-03-01

    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 Ca(2+) concentration ([Ca(2+)](i)) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca(2+)-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca(2+) 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 [Ca(2+)](i). Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca(2+) 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 [Ca(2+)](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

  7. Involvement of myosin light-chain kinase in endothelial cell retraction

    SciTech Connect

    Wysolmerski, R.B.; Lagunoff, D. )

    1990-01-01

    Permeabilized bovine pulmonary artery endothelial cell monolayers were used to investigate the mechanism of endothelial cell retraction. Postconfluent endothelial cells permeabilized with saponin retracted upon exposure to ATP and Ca{sup 2+}. Retraction was accompanied by thiophosphorylation of 19,000-Da myosin light chains when adenosine 5'-(gamma-({sup 35}S)thio)triphosphate was included in the medium. Both retraction and thiophosphorylation of myosin light chains exhibited a graded quantitative dependence on Ca{sup 2+}. When permeabilized monolayers were extracted in buffer D containing 100 mM KCl and 30 mM MgCl2 for 30 min, the cells failed to retract upon exposure to ATP and Ca{sup 2+}, and no thiophosphorylation of myosin light chains occurred. The ability both to retract and to thiophosphorylate myosin light chains was restored by the addition to the permeabilized, extracted cells of myosin light-chain kinase and calmodulin together but not by either alone. These studies indicate that endothelial cell retraction, as does smooth muscle contraction, depends on myosin light-chain kinase phosphorylation of myosin light chains.

  8. Regulation of Myosin II Dynamics by Phosphorylation and Dephosphorylation of Its Light Chain in Epithelial Cells

    PubMed Central

    Watanabe, Toshiyuki; Hosoya, Hiroshi

    2007-01-01

    Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase. PMID:17151359

  9. Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle

    PubMed Central

    Hong, Feng; Brizendine, Richard K.; Carter, Michael S.; Alcala, Diego B.; Brown, Avery E.; Chattin, Amy M.; Haldeman, Brian D.; Walsh, Michael P.; Facemyer, Kevin C.; Baker, Josh E.

    2015-01-01

    Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot–labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto–myosin and MLCK–myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting “stuck” on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle. PMID:26415568

  10. The C-terminal helix in subdomain 4 of the regulatory light chain is essential for myosin regulation.

    PubMed Central

    Rowe, T; Kendrick-Jones, J

    1993-01-01

    In vertebrate smooth/non-muscle myosins, phosphorylation of the regulatory light chains by a specific calmodulin-activated kinase controls both myosin head interaction with actin and assembly of the myosin into filaments. Previous studies have shown that the C-terminal domain of the regulatory light chain is crucial for the regulation of these myosin functions. To further dissect the role of this region of the light chain in myosin regulation, a series of chicken smooth muscle myosin regulatory light chain mutants has been constructed with successive C-terminal deletions. These mutants were synthesized in Escherichia coli and analysed by their ability to restore Ca2+ regulation to scallop myosin that had been stripped of its native regulatory light chains ('desensitized'). The results show that regulatory light chain mutants with deletions in the C-terminal helix in subdomain 4 were able to reform the regulatory Ca2+ binding site on the scallop myosin head, but had lost the ability to suppress scallop myosin filament assembly and interaction with actin in the absence of Ca2+. Further deletions in the C-terminal domain led to a gradual loss of ability to restore the regulatory Ca2+ binding site. Thus, the regions in the C-terminal half of the regulatory light chain responsible for myosin regulation can be identified. Images PMID:8223496

  11. Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis

    PubMed Central

    Valencia-Expósito, Andrea; Grosheva, Inna; Míguez, David G.; González-Reyes, Acaimo; Martín-Bermudo, María D.

    2016-01-01

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies. PMID:26888436

  12. Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis.

    PubMed

    Valencia-Expósito, Andrea; Grosheva, Inna; Míguez, David G; González-Reyes, Acaimo; Martín-Bermudo, María D

    2016-01-01

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies. PMID:26888436

  13. Sequence analysis of the myosin regulatory light chain gene of the vestimentiferan Riftia pachyptila.

    PubMed

    Ravaux, J; Hassanin, A; Deutsch, J; Gaill, F; Markmann-Mulisch, U

    2001-01-24

    We have isolated and characterized a cDNA (DNA complementary to RNA) clone (Rf69) from the vestimentiferan Riftia pachyptila. The cDNA insert consists of 1169 base pairs. The aminoacid sequence deduced from the longest reading frame is 193 residues in length, and clearly characterized it as a myosin regulatory light chain (RLC). The RLC primary structure is described in relation to its function in muscle contraction. The comparison with other RLCs suggested that Riftia myosin is probably regulated through its RLC either by phosphorylation like the vertebrate smooth muscle myosins, and/or by Ca2+-binding like the mollusk myosins. Riftia RLC possesses a N-terminal extension lacking in all other species besides the earthworm Lumbricus terrestris. Aminoacid sequence comparisons with a number of RLCs from vertebrates and invertebrates revealed a relatively high identity score (64%) between Riftia RLC and the homologous gene from Lumbricus. The relationships between the members of the myosin RLCs were examined by two phylogenetic methods, i.e. distance matrix and maximum parsimony. The resulting trees depict the grouping of the RLCs according to their role in myosin activity regulation. In all trees, Riftia RLC groups with RLCs that depend on Ca2+-binding for myosin activity regulation. PMID:11223252

  14. Atwood's Heavy Chain

    ERIC Educational Resources Information Center

    Beeken, Paul

    2011-01-01

    While perusing various websites in search of a more challenging lab for my students, I came across a number of ideas where replacing the string in an Atwood's machine with a simple ball chain like the kind found in lamp pulls created an interesting system to investigate. The replacement of the string produced a nice nonuniform acceleration, but…

  15. Crystal Structure of a Phosphorylated Light Chain Domain of Scallop Smooth-Muscle Myosin

    SciTech Connect

    Kumar, V.S.; Robinson, H.; O-Neall-Hennessey, E.; Reshetnikova, L.; Brown, J. H.; Szent-Gyorgyi, A. G.; Cohen, C.

    2011-11-02

    We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg{sup 16}, an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.

  16. Purification, Characterization and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkia.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in close species. In this study, MLC with the molecular mass of 18kDa was purified from crayfish (Procambarus clarkii) muscle fibrils. Its physicochemical characterization showed that the...

  17. Myosin light chain kinase facilitates endocytosis of synaptic vesicles at hippocampal boutons.

    PubMed

    Li, Lin; Wu, Xiaomei; Yue, Hai-Yuan; Zhu, Yong-Chuan; Xu, Jianhua

    2016-07-01

    At nerve terminals, endocytosis efficiently recycles vesicle membrane to maintain synaptic transmission under different levels of neuronal activity. Ca(2+) and its downstream signal pathways are critical for the activity-dependent regulation of endocytosis. An activity- and Ca(2+) -dependent kinase, myosin light chain kinase (MLCK) has been reported to regulate vesicle mobilization, vesicle cycling, and motility in different synapses, but whether it has a general contribution to regulation of endocytosis at nerve terminals remains unknown. We investigated this issue at rat hippocampal boutons by imaging vesicle endocytosis as the real-time retrieval of vesicular synaptophysin tagged with a pH-sensitive green fluorescence protein. We found that endocytosis induced by 200 action potentials (5-40 Hz) was slowed by acute inhibition of MLCK and down-regulation of MLCK with RNA interference, while the total amount of vesicle exocytosis and somatic Ca(2+) channel current did not change with MLCK down-regulation. Acute inhibition of myosin II similarly impaired endocytosis. Furthermore, down-regulation of MLCK prevented depolarization-induced phosphorylation of myosin light chain, an effect shared by blockers of Ca(2+) channels and calmodulin. These results suggest that MLCK facilitates vesicle endocytosis through activity-dependent phosphorylation of myosin downstream of Ca(2+) /calmodulin, probably as a widely existing mechanism among synapses. Our study suggests that MLCK is an important activity-dependent regulator of vesicle recycling in hippocampal neurons, which are critical for learning and memory. The kinetics of vesicle membrane endocytosis at nerve terminals has long been known to depend on activity and Ca(2+) . This study provides evidence suggesting that myosin light chain kinase increases endocytosis efficiency at hippocampal neurons by mediating Ca(2+) /calmodulin-dependent phosphorylation of myosin. The authors propose that this signal cascade may serve as

  18. Reconstitution of heavy chain and light chain 1 in cardiac subfragment-1 from hyperthyroid and euthyroid rabbit hearts.

    PubMed

    Ueda, S; Yamaoki, K; Nagai, R; Yazaki, Y

    1983-01-01

    It is now established that cardiac myosin from hyperthyroid rabbit hearts (TXM) exhibits high Ca2+ ATPase activity. The high Ca2+ ATPase activity of TXM was completely retained in cardiac myosin subfragment-1 (S-1) (1.33 +/- 0.04 mumol Pi/mg per min; euthyroid, 0.51 +/- 0.04). Cardiac S-1 from hyperthyroid and euthyroid rabbits (TXS-1 and NS-1) had the same pattern in SDS-polyacrylamide gel electrophoresis. The possible influence of heavy and light chains of TXM on increasing the ATPase activity was examined by reconstitution in the S-1 preparation. Crosswise reconstitution was performed using cardiac S-1 heavy chain (90,000 daltons) and light chain 1 (LC1) (27,000 daltons) from hyperthyroid and euthyroid hearts. Reconstitution was verified by using radiolabeled LC1. More than 95% of S-1 was recovered with full ATPase activity. When TXS-1 was reconstituted with LC1 from euthyroid hearts, the reconstituted molecule retained high ATPase activity. On the other hand, NS-1 reconstituted with LC1 from hyperthyroid hearts failed to increase the ATPase activity. The ATPase activity of S-1 was determined by the source of the heavy chain. These results suggest that the high Ca2+ ATPase activity of cardiac myosin and S-1 from hyperthyroid animals arises from the molecular alteration of the heavy chain induced by thyroxine administration. PMID:6304826

  19. Tumor Stiffness Is Unrelated to Myosin Light Chain Phosphorylation in Cancer Cells

    PubMed Central

    Fry, Madeline; Greene, Madelyne; Chernaya, Olga; Hu, Wen-Yang; Chew, Teng-Leong; Mahmud, Nadim; Kadkol, Shrihari S.; Glover, Sarah; Prins, Gail; Strakova, Zuzana; de Lanerolle, Primal

    2013-01-01

    Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors. PMID:24224004

  20. Molecular mechanisms of cardiomyopathy phenotypes associated with myosin light chain mutations.

    PubMed

    Huang, Wenrui; Szczesna-Cordary, Danuta

    2015-12-01

    We discuss here the potential mechanisms of action associated with hypertrophic (HCM) or dilated (DCM) cardiomyopathy causing mutations in the myosin regulatory (RLC) and essential (ELC) light chains. Specifically, we focus on four HCM mutations: RLC-A13T, RLC-K104E, ELC-A57G and ELC-M173V, and one DCM RLC-D94A mutation shown by population studies to cause different cardiomyopathy phenotypes in humans. Our studies indicate that RLC and ELC mutations lead to heart disease through different mechanisms with RLC mutations triggering alterations of the secondary structure of the RLC which further affect the structure and function of the lever arm domain and impose changes in the cross bridge cycling rates and myosin force generation ability. The ELC mutations exert their detrimental effects through changes in the interaction of the N-terminus of ELC with actin altering the cross talk between the thick and thin filaments and ultimately resulting in an altered force-pCa relationship. We also discuss the effect of mutations on myosin light chain phosphorylation. Exogenous myosin light chain phosphorylation and/or pseudo-phosphorylation were explored as potential rescue tools to treat hypertrophy-related cardiac phenotypes. PMID:26385864

  1. A Small-Molecule Inhibitor of T. gondii Motility Induces the Posttranslational Modification of Myosin Light Chain-1 and Inhibits Myosin Motor Activity

    PubMed Central

    Heaslip, Aoife T.; Leung, Jacqueline M.; Carey, Kimberly L.; Catti, Federica; Warshaw, David M.; Westwood, Nicholas J.; Ballif, Bryan A.; Ward, Gary E.

    2010-01-01

    Toxoplasma gondii is an obligate intracellular parasite that enters cells by a process of active penetration. Host cell penetration and parasite motility are driven by a myosin motor complex consisting of four known proteins: TgMyoA, an unconventional Class XIV myosin; TgMLC1, a myosin light chain; and two membrane-associated proteins, TgGAP45 and TgGAP50. Little is known about how the activity of the myosin motor complex is regulated. Here, we show that treatment of parasites with a recently identified small-molecule inhibitor of invasion and motility results in a rapid and irreversible change in the electrophoretic mobility of TgMLC1. While the precise nature of the TgMLC1 modification has not yet been established, it was mapped to the peptide Val46-Arg59. To determine if the TgMLC1 modification is responsible for the motility defect observed in parasites after compound treatment, the activity of myosin motor complexes from control and compound-treated parasites was compared in an in vitro motility assay. TgMyoA motor complexes containing the modified TgMLC1 showed significantly decreased motor activity compared to control complexes. This change in motor activity likely accounts for the motility defects seen in the parasites after compound treatment and provides the first evidence, in any species, that the mechanical activity of Class XIV myosins can be modulated by posttranslational modifications to their associated light chains. PMID:20084115

  2. Porcine myosin-VI: characterization of a new mammalian unconventional myosin

    PubMed Central

    1994-01-01

    We have cloned a new mammalian unconventional myosin, porcine myosin-VI from the proximal tubule cell line, LLC-PK1 (CL4). Porcine myosin-VI is highly homologous to Drosophila 95F myosin heavy chain, and together these two myosins comprise a sixth class of myosin motors. Myosin-VI exhibits ATP-sensitive actin-binding activities characteristic of myosins, and it is associated with a calmodulin light chain. Within LLC- PK1 cells, myosin-VI is soluble and does not associate with the major actin-containing domains. Within the kidney, however, myosin-VI is associated with sedimentable structures and specifically locates to the actin- and membrane-rich apical brush border domain of the proximal tubule cells. This motor was not enriched within the glomerulus, capillaries, or distal tubules. Myosin-VI associates with the proximal tubule cytoskeleton in an ATP-sensitive fashion, suggesting that this motor is associated with the actin cytoskeleton within the proximal tubule cells. Given the difference in association of myosin-VI with the apical cytoskeleton between LLC-PK1 cells and adult kidney, it is likely that this cell line does not fully differentiate to form functional proximal tubule cells. Myosin-VI may require the presence of additional elements, only found in vivo in proximal tubule cells, to properly locate to the apical domain. PMID:7929586

  3. Heterogeneity of myofibrillar proteins in lobster fast and slow muscles: variants of troponin, paramyosin, and myosin light chains comprise four distinct protein assemblages

    SciTech Connect

    Mykles, D.L.

    1985-01-01

    Fast and slow muscles from the claws and abdomen of the American lobster Homarus americanus were examined for adenosine triphosphatase (ATPase) activity and for differences in myofibrillar proteins. Both myosin and actomyosin ATPase were correlated with fiber composition and contractile speed. Four distinct patterns of myofibrilla proteins observed in sodium dodecyl sulfate-polyacrylamide gels were distinguished by different assemblages of regulatory and contractile protein variants. A total of three species of troponin-T, five species of troponin-I, and three species of troponin-C were observed. Lobster myosins contained two groups of light chains (LC), termed alpha and beta. There were three ..cap alpha..-LC variants and two ..beta..-LC variants. There were no apparent differences in myosin heavy chain, actin, and tropomyosin. Only paramyosin showed a pattern completely consistent with muscle fiber type: slow fibers contained a species (105 kD) slightly smaller than the principle variant (110 kD) in fast fibers. It is proposed that the type of paramyosin present could provide a biochemical marker to identify the fiber composition of muscles that have not been fully characterized. The diversity of troponin and myosin LC variants suggests that subtle differences in physiological performance exist within the broader categories of fast- and slow-twitch muscles. 31 references, 6 figures, 2 tables.

  4. Myosin light chain kinase regulates cell polarization independently of membrane tension or Rho kinase

    PubMed Central

    Lou, Sunny S.; Diz-Muñoz, Alba; Weiner, Orion D.; Fletcher, Daniel A.

    2015-01-01

    Cells polarize to a single front and rear to achieve rapid actin-based motility, but the mechanisms preventing the formation of multiple fronts are unclear. We developed embryonic zebrafish keratocytes as a model system for investigating establishment of a single axis. We observed that, although keratocytes from 2 d postfertilization (dpf) embryos resembled canonical fan-shaped keratocytes, keratocytes from 4 dpf embryos often formed multiple protrusions despite unchanged membrane tension. Using genomic, genetic, and pharmacological approaches, we determined that the multiple-protrusion phenotype was primarily due to increased myosin light chain kinase (MLCK) expression. MLCK activity influences cell polarity by increasing myosin accumulation in lamellipodia, which locally decreases protrusion lifetime, limiting lamellipodial size and allowing for multiple protrusions to coexist within the context of membrane tension limiting protrusion globally. In contrast, Rho kinase (ROCK) regulates myosin accumulation at the cell rear and does not determine protrusion size. These results suggest a novel MLCK-specific mechanism for controlling cell polarity via regulation of myosin activity in protrusions. PMID:25918227

  5. Two Cases of Heavy Chain MGUS.

    PubMed

    Van Keer, Jan; Meijers, Björn; Delforge, Michel; Verhoef, Gregor; Poesen, Koen

    2016-01-01

    Heavy chain diseases are rare variants of B-cell lymphomas that produce one of three classes of immunoglobulin heavy chains, without corresponding light chains. We describe two patients with asymptomatic heavy chain monoclonal gammopathy. The first patient is a 51-year-old woman with alpha paraprotein on serum immunofixation. The second case is a 46-year-old woman with gamma paraprotein on urine immunofixation. Neither patient had corresponding monoclonal light chains. Workup for multiple myeloma and lymphoma was negative in both patients. These two cases illustrate that heavy chain monoclonal gammopathy can exist in the absence of clinically apparent malignancy. Only a few reports of "heavy chain MGUS" have been described before. In the absence of specialized guidelines, we suggest a similar follow-up as for MGUS, while taking into account the higher probability of progression to lymphoma than to myeloma. PMID:27213064

  6. Two Cases of Heavy Chain MGUS

    PubMed Central

    Meijers, Björn; Delforge, Michel; Verhoef, Gregor; Poesen, Koen

    2016-01-01

    Heavy chain diseases are rare variants of B-cell lymphomas that produce one of three classes of immunoglobulin heavy chains, without corresponding light chains. We describe two patients with asymptomatic heavy chain monoclonal gammopathy. The first patient is a 51-year-old woman with alpha paraprotein on serum immunofixation. The second case is a 46-year-old woman with gamma paraprotein on urine immunofixation. Neither patient had corresponding monoclonal light chains. Workup for multiple myeloma and lymphoma was negative in both patients. These two cases illustrate that heavy chain monoclonal gammopathy can exist in the absence of clinically apparent malignancy. Only a few reports of “heavy chain MGUS” have been described before. In the absence of specialized guidelines, we suggest a similar follow-up as for MGUS, while taking into account the higher probability of progression to lymphoma than to myeloma. PMID:27213064

  7. Cooperative Regulation of Myosin-Actin Interactions by a Continuous Flexible Chain I: Actin-Tropomyosin Systems

    PubMed Central

    Smith, D. A.; Maytum, R.; Geeves, M. A.

    2003-01-01

    We present a model for cooperative myosin binding to the regulated actin filament, where tropomyosins are treated as a weakly-confined continuous flexible chain covering myosin binding sites. Thermal fluctuations in chain orientation are initially required for myosin binding, leaving kinked regions under which subsequent myosins may bind without further distortion of the chain. Statistical mechanics predicts the fraction of sites with bound myosin-S1 as a function of their affinities. Published S1 binding curves to regulated filaments with different tropomyosin isoforms are fitted by varying the binding constant, chain persistence length ν (in actin monomers), and chain kink energy A from a single bound S1. With skeletal tropomyosin, we find an S1 actin-binding constant of 2.2 × 107 M−1, A = 1.6 kBT and ν = 2.7. Similar persistence lengths are found with yeast tropomyosin. Larger values are found for tropomyosin-troponin in the presence of calcium (ν = 3.7) and tropomyosins from smooth muscle and fibroblasts (ν = 4.5). The relationship of these results to structural information and the rigid-unit model of McKillop and Geeves is discussed. PMID:12719245

  8. Distinct tissue distributions and subcellular localizations of differently phosphorylated forms of the myosin regulatory light chain in Drosophila.

    PubMed

    Zhang, Liang; Ward, Robert E

    2011-01-01

    Nonmuscle myosin II (myosin hereafter) has well-established roles in generating contractile force on actin filaments during morphogenetic processes in all metazoans. Myosin activation is regulated by phosphorylation of the myosin regulatory light chain (MRLC, encoded by spaghettisquash or sqh in Drosophila) first on Ser21 and subsequently on Thr20. These phosphorylation events are positively controlled by a variety of kinases including myosin light chain kinase, Rho kinase, citron kinase, and AMP kinase and are negatively regulated by myosin phosphatase. The activation of myosin is thus highly regulated and likely developmentally controlled. In order to monitor the activity of myosin during development, we have generated antibodies against the monophosphorylated (Sqh1P) and diphosphorylated (Sqh2P) forms of Sqh. We first show that the antibodies are highly specific. We then used these antibodies to monitor myosin activation in wild type Drosophila tissues. Interestingly, Sqh1P and Sqh2P show distinct patterns of expression in embryos. Sqh1P is expressed nearly ubiquitously and outlines cells consistent with a junctional localization, whereas Sqh2P is strongly expressed on the apical surfaces and in filopodia of tissues undergoing extensive cell shape change or cell movements including the invaginating fore- and hindgut, the invaginating tracheal system, the dorsal pouch and the dorsal most row of epidermal (DME) cells during dorsal closure. In imaginal discs, Sqh1P predominantly localizes in the adherens junction, whereas Sqh2P locates to the apical domain. These antibodies thus have the potential to be very useful in monitoring myosin activation for functional studies of morphogenesis in Drosophila. PMID:20920606

  9. Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin

    SciTech Connect

    Vu, N.D.; Wagner, P.D.

    1987-07-28

    Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of /sup 32/P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca/sup 2 +/- and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1. Although this subfragment 1 contained intact light chains, its actin-activated ATPase activity was not affected by light chain phosphorylation.

  10. Estradiol modulates myosin regulatory light chain phosphorylation and contractility in skeletal muscle of female mice.

    PubMed

    Lai, Shaojuan; Collins, Brittany C; Colson, Brett A; Kararigas, Georgios; Lowe, Dawn A

    2016-05-01

    Impairment of skeletal muscle function has been associated with changes in ovarian hormones, especially estradiol. To elucidate mechanisms of estradiol on skeletal muscle strength, the hormone's effects on phosphorylation of the myosin regulatory light chain (pRLC) and muscle contractility were investigated, hypothesizing an estradiol-specific beneficial impact. In a skeletal muscle cell line, C2C12, pRLC was increased by 17β-estradiol (E2) in a concentration-dependent manner. In skeletal muscles of C57BL/6 mice that were E2 deficient via ovariectomy (OVX), pRLC was lower than that from ovary-intact, sham-operated mice (Sham). The reduced pRLC in OVX muscle was reversed by in vivo E2 treatment. Posttetanic potentiation (PTP) of muscle from OVX mice was low compared with that from Sham mice, and this decrement was reversed by acute E2 treatment, demonstrating physiological consequence. Western blot of those muscles revealed that low PTP corresponded with low pRLC and higher PTP with greater pRLC. We aimed to elucidate signaling pathways affecting E2-mediated pRLC using a kinase inhibitor library and C2C12 cells as well as a specific myosin light chain kinase inhibitor in muscles. PI3K/Akt, MAPK, and CamKII were identified as candidate kinases sensitive to E2 in terms of phosphorylating RLC. Applying siRNA strategy in C2C12 cells, pRLC triggered by E2 was found to be mediated by estrogen receptor-β and the G protein-coupled estrogen receptor. Together, these results provide evidence that E2 modulates myosin pRLC in skeletal muscle and is one mechanism by which this hormone can affect muscle contractility in females. PMID:26956186

  11. MODULAR STRUCTURE OF SMOOTH MUSCLE MYOSIN LIGHT CHAIN KINASE: HYDRODYNAMIC MODELING AND FUNCTIONAL IMPLICATIONS†

    PubMed Central

    Mabuchi, Yasuko; Mabuchi, Katsuhide; Stafford, Walter F.; Grabarek, Zenon

    2010-01-01

    Smooth muscle myosin light chain kinase (smMLCK) is a calcium/calmodulin dependent enzyme that activates contraction of smooth muscle. The polypeptide chain of rabbit uterine smMLCK (Swiss-Prot: P29294) contains the catalytic/regulatory domain, three immunoglobulin related motifs (Ig), one fibronectin related motif (Fn3), a repetitive, proline rich segment (PEVK) and, at the N-terminus, a unique F-actin binding domain. We have evaluated the spatial arrangement of these domains in a recombinant 125 kDa full-length smMLCK and its two catalytically active C-terminal fragments (77 kDa, residues 461-1147 and 61 kDa, residues 461-1002). Electron microscopic images of smMLCK cross-linked to F-actin show particles at variable distance (11-55 nm) from the filament, suggesting that a well-structured C-terminal segment of smMLCK is connected to the actin-binding domain by a long, flexible tether. We have used structural homology and molecular dynamics methods to construct various all-atom representation models of smMLCK and its two fragments. The theoretical sedimentation coefficients computed with the program HYDROPRO were compared with those determined by sedimentation velocity. We found agreement between the predicted and observed sedimentation coefficients for models in which the independently folded catalytic domain, Fn3 and Ig domains are aligned consecutively on the long axis of the molecule. The PEVK segment is modeled as an extensible linker that enables smMLCK to remain bound to F-actin and simultaneously activate the myosin heads of adjacent myosin filaments at a distance of 40 nm or more. The structural properties of smMLCK may contribute to the elasticity of smooth muscle cells. PMID:20196616

  12. Interplay between the effects of a Protein Kinase C phosphomimic (T204E) and a dilated cardiomyopathy mutation (K211Δ or R206W) in rat cardiac troponin T blunts the magnitude of muscle length-mediated crossbridge recruitment against the β-myosin heavy chain background.

    PubMed

    Michael, John Jeshurun; Gollapudi, Sampath K; Chandra, Murali

    2016-06-01

    Failing hearts of dilated cardiomyopathy (DCM)-patients reveal systolic dysfunction and upregulation of several Protein Kinase C (PKC) isoforms. Recently, we demonstrated that the functional effects of T204E, a PKC phosphomimic of cardiac troponin T (TnT), were differently modulated by α- and β-myosin heavy chain (MHC) isoforms. Therefore, we hypothesized that the interplay between the effects of T204E and a DCM-linked mutation (K211Δ or R206W) in TnT would modulate contractile parameters linked-to systolic function in an MHC-dependent manner. To test our hypothesis, five TnT variants (wildtype, K211Δ, K211Δ + T204E, R206W, and R206W + T204E) were generated and individually reconstituted into demembranated cardiac muscle fibers from normal (α-MHC) and propylthiouracil-treated (β-MHC) rats. Steady-state and mechano-dynamic measurements were performed on reconstituted fibers. Myofilament Ca(2+) sensitivity (pCa50) was decreased by both K211Δ and R206W to a greater extent in α-MHC fibers (~0.15 pCa units) than in β-MHC fibers (~0.06 pCa units). However, T204E exacerbated the attenuating influence of both mutants on pCa50 only in β-MHC fibers. Moreover, the magnitude of muscle length (ML)-mediated crossbridge (XB) recruitment was decreased by K211Δ + T204E (~47 %), R206W (~34 %), and R206W + T204E (~36 %) only in β-MHC fibers. In relevance to human hearts, which predominantly express β-MHC, our data suggest that the interplay between the effects of DCM mutations, PKC phosphomimic in TnT, and β-MHC lead to systolic dysfunction by attenuating pCa50 and the magnitude of ML-mediated XB recruitment. PMID:27411801

  13. Myosin Light-Chain Kinase Is Necessary for Membrane Homeostasis in Cochlear Inner Hair Cells

    PubMed Central

    Chen, Chen; Xu, Lin; Zhang, Wen-Cheng; Fan, Chi; Peng, Ya-Jing; Chen, Jie; He, Wei-Qi; Guo, Shi-Ying; Zuo, Jian; Gao, Xia; Zhu, Min-Sheng

    2012-01-01

    The structural homeostasis of the cochlear hair cell membrane is critical for all aspects of sensory transduction, but the regulation of its maintenance is not well understood. In this report, we analyzed the cochlear hair cells of mice with specific deletion of myosin light chain kinase (MLCK) in inner hair cells. MLCK-deficient mice showed impaired hearing, with a 5- to 14-dB rise in the auditory brainstem response (ABR) thresholds to clicks and tones of different frequencies and a significant decrease in the amplitude of the ABR waves. The mutant inner hair cells produced several ball-like structures around the hair bundles in vivo, indicating impaired membrane stability. Inner hair cells isolated from the knockout mice consistently displayed less resistance to hypoosmotic solution and less membrane F-actin. Myosin light-chain phosphorylation was also reduced in the mutated inner hair cells. Our results suggest that MLCK is necessary for maintaining the membrane stability of inner hair cells. PMID:22485190

  14. Ischemia/reperfusion-induced myosin light chain 1 phosphorylation increases its degradation by matrix metalloproteinase-2

    PubMed Central

    Cadete, Virgilio J. J.; Sawicka, Jolanta; Jaswal, Jagdip; Lopaschuk, Gary D.; Schulz, Richard; Szczesna-Cordary, Danuta; Sawicki, Grzegorz

    2012-01-01

    Summary Degradation of myosin light chain 1 (MLC1) by matrix metalloproteinase-2 (MMP-2) during myocardial ischemia/reperfusion (I/R) injury has been established. However, the exact mechanisms controlling this process remain unknown. I/R increases the phosphorylation of MLC1, but the consequences of this modification are not known. We hypothesized that phosphorylation of MLC1 plays an important role in its degradation by MMP-2. To examine this, isolated perfused rat hearts were subjected to 20 min global ischemia followed by 30 min of aerobic reperfusion. I/R increased phosphorylation of MLC1 (as measured by mass spectrometry). If hearts were subjected to I/R in the presence of ML-7 (a myosin light chain kinase (MLCK) inhibitor) or doxycycline (a MMP inhibitor) an improved recovery of contractile function was seen compared to aerobic hearts and MLC1 was protected from degradation. Enzyme kinetic studies revealed an increased affinity of MMP-2 for the phosphorylated form of MLC1 compared to non-phosphorylated MLC1. We conclude that MLC1 phosphorylation is important mechanism controlling the intracellular action of MMP-2 and promoting the degradation of MLC1. These results further support previous findings implicating posttranslational modifications of contractile proteins as a key factor in the pathology of cardiac dysfunction during and following ischemia. PMID:22564771

  15. Planarian myosin essential light chain is involved in the formation of brain lateral branches during regeneration.

    PubMed

    Yu, Shuying; Chen, Xuhui; Yuan, Zuoqing; Zhou, Luming; Pang, Qiuxiang; Mao, Bingyu; Zhao, Bosheng

    2015-08-01

    The myosin essential light chain (ELC) is a structure component of the actomyosin cross-bridge, however, the functions in the central nervous system (CNS) development and regeneration remain poorly understood. Planarian Dugesia japonica has revealed fundamental mechanisms and unique aspects of neuroscience and neuroregeneration. In this study, the cDNA DjElc, encoding a planarian essential light chain of myosin, was identified from the planarian Dugesia japonica cDNA library. It encodes a deduced protein with highly conserved functionally domains EF-Hand and Ca(2+) binding sites that shares significant similarity with other members of ELC. Whole mount in situ hybridization studies show that DjElc expressed in CNS during embryonic development and regeneration of adult planarians. Loss of function of DjElc by RNA interference during planarian regeneration inhibits brain lateral branches regeneration completely. In conclusion, these results demonstrated that DjElc is required for maintenance of neurons and neurite outgrowth, particularly for involving the brain later branch regeneration. PMID:25585662

  16. Myosin light chain phosphorylation during the contraction cycle of frog muscle.

    PubMed

    Bárány, K; Bárány, M; Gillis, J M; Kushmerick, M J

    1980-04-01

    Changes in the [32P]phosphate content of proteins during contraction were investigated with sartorius and semitendinosus muscles dissected from live frogs injected with [32P]orthophosphate. During a single tetanus, the only significant change was the increase in the [32P]phosphate content of the 18,000-dalton light chain of myosin. The extent of light chain phosphorylation was a function of stimulus duration and it amounted maximally to 0.35 mol of [32P]phosphate transferred per mol of light chain. The extent of phosphorylation in stimulated and stretched semitendinosus muscles, which were unable to produce active tension, was nearly identical to that in muscles stimulated at standard rest length, when the time of stimulation was over a half-second. Maximal light chain phosphorylation was also observed in muscles treated with caffein. These results provide evidence for the activation of the light chain kinase in the intact muscle through a process involving Ca2+. The phosphorylation of the light chain associated with tetanic stimulation was reversible. After short tetanuses, dephosphorylation of light chain approximately followed relaxation and after longer tetanuses, dephosphorylation lagged behind relaxation. The role of light chain phosphorylation was investigated in caffeine-treated and untreated muscles by measuring the Ca content of actin and the [32P]phosphate content of light chain. Phosphorylation of light chain protected the actin-bound Ca against removal by EDTA stoichiometrically. It is postulated that the physiological role of light chain phosphorylation is to increase the rate of combination of the cross-bridges with the actin filaments in the contracting phase of the mechanical activity. PMID:7364050

  17. Myosin Heavy Chain Composition of the Human Genioglossus Muscle

    ERIC Educational Resources Information Center

    Daugherty, Megan; Luo, Qingwei; Sokoloff, Alan J.

    2012-01-01

    Background: The human tongue muscle genioglossus (GG) is active in speech, swallowing, respiration, and oral transport, behaviors encompassing a wide range of tongue shapes and movement speeds. Studies demonstrate substantial diversity in patterns of human GG motor unit activation, but whether this is accompanied by complex expression of muscle…

  18. The role of the N-terminus of the myosin essential light chain in cardiac muscle contraction

    PubMed Central

    Kazmierczak, Katarzyna; Xu, Yuanyuan; Jones, Michelle; Guzman, Georgianna; Hernandez, Olga M.; Kerrick, W. Glenn L.; Szczesna-Cordary, Danuta

    2011-01-01

    Summary 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 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 non-pathologic 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. PMID:19361417

  19. Essential myosin light chain as a target for caspase-3 in failing myocardium

    PubMed Central

    Moretti, Alessandra; Weig, Hans-Jörg; Ott, Thomas; Seyfarth, Melchior; Holthoff, Hans-Peter; Grewe, Diana; Gillitzer, Angelika; Bott-Flügel, Lorenz; Schömig, Albert; Ungerer, Martin; Laugwitz, Karl-Ludwig

    2002-01-01

    Programmed cell death involves the activation of caspase proteases that can mediate the cleavage of vital cytoskeletal proteins. We have recently reported that, in failing cardiac myocytes, caspase-3 activation is associated with a reduction in contractile performance. In this study we used a modified yeast two-hybrid system to screen for caspase-3 interacting proteins of the cardiac cytoskeleton. We identified ventricular essential myosin light chain (vMLC1) as a target for caspase-3. By sequencing and site-directed mutagenesis, a noncanonical cleavage site for caspase-3 was mapped to the C-terminal DFVE135G motif. We demonstrated that vMLC1 cleavage in failing myocardium in vivo is associated with a morphological disruption of the organized vMLC1 staining of sarcomeres, and with a reduction in myocyte contractile performance. Adenoviral gene transfer of the caspase inhibitor p35 in vivo prevented caspase-3 activation and vMLC1 cleavage, with positive impact on contractility. These data suggest that direct cleavage of vMLC1 by activated caspase-3 may contribute to depression of myocyte function by altering cross-bridge interaction between myosin and actin molecules. Therefore, activation of apoptotic pathways in the heart may lead to contractile dysfunction before cell death. PMID:12186978

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

    PubMed Central

    Muthu, Priya; Wang, Li; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Huang, Wenrui; Hernandez, Olga M.; Kawai, Masataka; Irving, Thomas C.; Szczesna-Cordary, Danuta

    2011-01-01

    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 I1,1/I1,0, 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.—Muthu, P., Wang, L., Yuan, C.-C., Kazmierczak, K., Huang, W., Hernandez, O. M., Kawai, M., Irving, T. C., Szczesna-Cordary, D. Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction. PMID:21885653

  1. Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities.

    PubMed

    Chang, Audrey N; Mahajan, Pravin; Knapp, Stefan; Barton, Hannah; Sweeney, H Lee; Kamm, Kristine E; Stull, James T

    2016-07-01

    The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties. PMID:27325775

  2. Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells

    PubMed Central

    Das, Provas; Saha, Shekhar; Chandra, Sunandini; Das, Alakesh; Dey, Sumit K.; Das, Mahua R.; Sen, Shamik; Sarkar, Debi P.; Jana, Siddhartha S.

    2015-01-01

    Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (−) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion. PMID:25993465

  3. Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells.

    PubMed

    Das, Provas; Saha, Shekhar; Chandra, Sunandini; Das, Alakesh; Dey, Sumit K; Das, Mahua R; Sen, Shamik; Sarkar, Debi P; Jana, Siddhartha S

    2015-01-01

    Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (-) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion. PMID:25993465

  4. Papaverine Prevents Vasospasm by Regulation of Myosin Light Chain Phosphorylation and Actin Polymerization in Human Saphenous Vein

    PubMed Central

    Hocking, Kyle M.; Putumbaka, Gowthami; Wise, Eric S.; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

    2016-01-01

    Objective Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. Approach and Results HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. Conclusions These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm. PMID:27136356

  5. Orientation of spin-labeled light chain-2 exchanged onto myosin cross-bridges in glycerinated muscle fibers.

    PubMed Central

    Hambly, B; Franks, K; Cooke, R

    1991-01-01

    Electron paramagnetic resonance (EPR) spectroscopy has been used to study the angular distribution of a spin label attached to rabbit skeletal muscle myosin light chain 2. A cysteine reactive spin label, 3-(5-fluoro-2,4-dinitroanilino)-2,2,5,5- tetramethyl-1-pyrrolidinyloxy (FDNA-SL) was bound to purified LC2. The labeled LC2 was exchanged into glycerinated muscle fibers and into myosin and its subfragments. Analysis of the spectra of labeled fibers in rigor showed that the probe was oriented with respect to the fiber axis, but that it was also undergoing restricted rotations. The motion of the probe could be modeled assuming rapid rotational diffusion (rotational correlation time faster than 5 ns) within a "cone" whose full width was 70 degrees. Very different spectra of rigor fibers were obtained with the fiber oriented parallel and perpendicular to the magnetic field, showing that the centroid of each cone had the same orientation for all myosin heads, making an angle of approximately 74 degrees to the fiber axis. Binding of light chains or labeled myosin subfragment-1 to ion exchange heads immobilized the probes, showing that most of the motion of the probe arose from protein mobility and not from mobility of the probe relative to the protein. Relaxed labeled fibers produced EPR spectra with a highly disordered angular distribution, consistent with myosin heads being detached from the thin filament and undergoing large angular motions. Addition of pyrophosphate, ADP, or an ATP analogue (AMPPNP), in low ionic strength buffer where these ligands do not dissociate cross-bridges from actin, failed to perturb the rigor spectrum. Applying static strains as high as 0.16 N/mm2 to the labeled rigor fibers also failed to change the orientation of the spin label. Labeled light chain was exchanged into myosin subfragment-1 (S1) and the labeled S1 was diffused into fibers. EPR spectra of these fibers had a component similar to that seen in the spectra of fibers into which

  6. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon.

    PubMed

    Del Coso, Juan; Valero, Marjorie; Lara, Beatriz; Salinero, Juan José; Gallo-Salazar, César; Areces, Francisco

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin producing increases in force development during skeletal muscle contraction. It has been suggested that MLCK gene polymorphisms might alter RLC phosphorylation thereby decreasing the ability to produce force and to resist strain during voluntary muscle contractions. Thus, the genetic variations in the MLCK gene might predispose some individuals to higher values of muscle damage during exercise, especially during endurance competitions. The aim of this investigation was to determine the influence of MLCK genetic variants on exercise-induced muscle damage produced during a marathon. Sixty-seven experienced runners competed in a marathon race. The MLCK genotype (C37885A) of these marathoners was determined. Before and after the race, a sample of venous blood was obtained to assess changes in serum myoglobin concentrations and leg muscle power changes were measured during a countermovement jump. Self-reported leg muscle pain and fatigue were determined by questionnaires. A total of 59 marathoners (88.1%) were CC homozygotes and 8 marathoners (11.9%) were CA heterozygotes. The two groups of participants completed the race with a similar time (228 ± 33 vs 234 ± 39 min; P = 0.30) and similar self-reported values for fatigue (15 ± 2 vs 16 ± 2 A.U.; P = 0.21) and lower-limb muscle pain (6.2 ± 1.7 vs 6.6 ± 1.8 cm; P = 0.29). However, CC marathoners presented higher serum myoglobin concentrations (739 ± 792 vs 348 ± 144 μg·mL-1; P = 0.03) and greater pre-to-post- race leg muscle power reduction (-32.7 ± 15.7 vs -21.2 ± 21.6%; P = 0.05) than CA marathoners. CA heterozygotes for MLCK C37885A might present higher exercise-induced muscle damage after a marathon competition than CC counterparts. PMID:27483374

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

    PubMed

    Yuan, Chen-Ching; Muthu, Priya; Kazmierczak, Katarzyna; Liang, Jingsheng; Huang, Wenrui; Irving, Thomas C; Kanashiro-Takeuchi, Rosemeire M; Hare, Joshua M; Szczesna-Cordary, Danuta

    2015-07-28

    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 Ca(2+) 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. PMID:26124132

  8. Myosin Light Chain Kinase (MLCK) Gene Influences Exercise Induced Muscle Damage during a Competitive Marathon

    PubMed Central

    Valero, Marjorie; Lara, Beatriz; Salinero, Juan José; Gallo-Salazar, César; Areces, Francisco

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin producing increases in force development during skeletal muscle contraction. It has been suggested that MLCK gene polymorphisms might alter RLC phosphorylation thereby decreasing the ability to produce force and to resist strain during voluntary muscle contractions. Thus, the genetic variations in the MLCK gene might predispose some individuals to higher values of muscle damage during exercise, especially during endurance competitions. The aim of this investigation was to determine the influence of MLCK genetic variants on exercise-induced muscle damage produced during a marathon. Sixty-seven experienced runners competed in a marathon race. The MLCK genotype (C37885A) of these marathoners was determined. Before and after the race, a sample of venous blood was obtained to assess changes in serum myoglobin concentrations and leg muscle power changes were measured during a countermovement jump. Self-reported leg muscle pain and fatigue were determined by questionnaires. A total of 59 marathoners (88.1%) were CC homozygotes and 8 marathoners (11.9%) were CA heterozygotes. The two groups of participants completed the race with a similar time (228 ± 33 vs 234 ± 39 min; P = 0.30) and similar self-reported values for fatigue (15 ± 2 vs 16 ± 2 A.U.; P = 0.21) and lower-limb muscle pain (6.2 ± 1.7 vs 6.6 ± 1.8 cm; P = 0.29). However, CC marathoners presented higher serum myoglobin concentrations (739 ± 792 vs 348 ± 144 μg·mL-1; P = 0.03) and greater pre-to-post- race leg muscle power reduction (-32.7 ± 15.7 vs -21.2 ± 21.6%; P = 0.05) than CA marathoners. CA heterozygotes for MLCK C37885A might present higher exercise-induced muscle damage after a marathon competition than CC counterparts. PMID:27483374

  9. Light chain-dependent myosin structural dynamics in solution investigated by transient electrical birefringence.

    PubMed Central

    Eden, D; Highsmith, S

    1997-01-01

    The technique of transient electrical birefringence was used to compare some of the electric and structural dynamic properties of myosin subfragment 1 (S1(elc, rlc)), which has both the essential and regulatory light chains bound, to S1(elc), which has only an essential light chain. The rates of rotational Brownian motion indicate that S1(elc, rlc) is larger, as expected. The permanent electric dipole moment of S1(elc, rlc) is also larger, indicating that the regulatory light chain portion of S1(elc, rlc) has a dipole moment and that it is aligned head-to-tail with the dipole moment of the S1(elc) portion. The permanent electric dipoles decrease with increasing ionic strength, apparently because of ion binding to surface charges. Both S1(elc, rlc) and S1(elc) have intrinsic segmental flexibility, as detected by the ability to selectively align segments with a brief weak electric field. However, unlike S1(elc), which can be structurally distorted by the action of a brief strong electric field, S1(elc, rlc) is stiffer and cannot be distorted by fields as high as 7800 V/cm applied to its approximately 8000 D permanent electric dipole moment. The S1 . MgADP . Pi analog S1 . MgADP . Vi is smaller than S1 . MgADP, for both S1(elc, rlc) and S1(elc). Interestingly, the smaller, stiffer S1(elc, rlc) . MgADP . Vi complex retains intrinsic segmental flexibility. These results are discussed within a framework of current hypotheses of force-producing mechanisms that involve S1 segmental motion and/or the loss of cross-bridge flexibility during force production. PMID:9251811

  10. Orientation of the N- and C-terminal lobes of the myosin regulatory light chain in cardiac muscle.

    PubMed

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm

    2015-01-20

    The orientations of the N- and C-terminal lobes of the cardiac isoform of the myosin regulatory light chain (cRLC) in the fully dephosphorylated state in ventricular trabeculae from rat heart were determined using polarized fluorescence from bifunctional sulforhodamine probes. cRLC mutants with one of eight pairs of surface-accessible cysteines were expressed, labeled with bifunctional sulforhodamine, and exchanged into demembranated trabeculae to replace some of the native cRLC. Polarized fluorescence data from the probes in each lobe were combined with RLC crystal structures to calculate the lobe orientation distribution with respect to the filament axis. The orientation distribution of the N-lobe had three distinct peaks (N1-N3) at similar angles in relaxation, isometric contraction, and rigor. The orientation distribution of the C-lobe had four peaks (C1-C4) in relaxation and isometric contraction, but only two of these (C2 and C4) remained in rigor. The N3 and C4 orientations are close to those of the corresponding RLC lobes in myosin head fragments bound to isolated actin filaments in the absence of ATP (in rigor), but also close to those of the pair of heads folded back against the filament surface in isolated thick filaments in the so-called J-motif conformation. The N1 and C1 orientations are close to those expected for actin-bound myosin heads with their light chain domains in a pre-powerstroke conformation. The N2 and C3 orientations have not been observed previously. The results show that the average change in orientation of the RLC region of the myosin heads on activation of cardiac muscle is small; the RLC regions of most heads remain in the same conformation as in relaxation. This suggests that the orientation of the dephosphorylated RLC region of myosin heads in cardiac muscle is primarily determined by an interaction with the thick filament surface. PMID:25606679

  11. Myosin light chain phosphorylation in sup 32 P-labeled rabbit aorta stimulated by phorbol 12,13-dibutyrate and phenylephrine

    SciTech Connect

    Singer, H.A.; Oren, J.W.; Benscoter, H.A. )

    1989-12-15

    The mechanism(s) of force development in vascular smooth muscle following pharmacological activation of protein kinase C by phorbol esters are not known. In this study, we examined the myosin light chain phosphorylation response following stimulation by phorbol 12,13-dibutyrate (PDB) or phenylephrine in rabbit aorta which had been incubated with 32PO4 in order to label ATP pools. Through tryptic phosphopeptide mapping of myosin light chain from intact tissue and comparison to controls using purified components, we inferred that Ca2+-dependent force stimulated by PDB was associated with small increases in serine-19 phosphorylation, consistent with a contractile mechanism involving indirect activation of myosin light chain kinase. Additional residues, consistent with the in vitro substrate specificity of protein kinase C, were also observed to be phosphorylated in response to PDB and represented proportionately a larger fraction of the total phosphorylated myosin light chain in Ca2+-depleted tissues. Stimulation by an alpha 1-adrenergic agonist (phenylephrine) resulted in phosphorylation of residues which were consistent with an activation mechanism involving myosin light chain kinase only. These results indicate that in rabbit aorta the contractile effects of PDB may be partially mediated by Ca2+-dependent activation of myosin light chain kinase. However, the data do not rule out a component of the PDB-stimulated contractile response which is independent of myosin light chain phosphorylation on the serine-19 residue. In addition, activation by a more physiological stimulus, phenylephrine, does not result in protein kinase C-mediated myosin light chain phosphorylation.

  12. Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury

    PubMed Central

    Wang, Ting; Mathew, Biji; Wu, Xiaomin; Shimizu, Yuka; Rizzo, Alicia N.; Dudek, Steven M.; Weichselbaum, Ralph R.; Jacobson, Jeffrey R.; Hecker, Louise

    2016-01-01

    Abstract Radiotherapy as a primary treatment for thoracic malignancies induces deleterious effects, such as acute or subacute radiation-induced lung injury (RILI). Although the molecular etiology of RILI is controversial and likely multifactorial, a potentially important cellular target is the lung endothelial cytoskeleton that regulates paracellular gap formation and the influx of macromolecules and fluid to the alveolar space. Here we investigate the central role of a key endothelial cytoskeletal regulatory protein, the nonmuscle isoform of myosin light chain kinase (nmMLCK), in an established murine RILI model. Our results indicate that thoracic irradiation significantly augmented nmMLCK protein expression and enzymatic activity in murine lungs. Furthermore, genetically engineered mice harboring a deletion of the nmMLCK gene (nmMLCK−/− mice) exhibited protection from RILI, as assessed by attenuated vascular leakage and leukocyte infiltration. In addition, irradiated wild-type mice treated with two distinct MLCK enzymatic inhibitors, ML-7 and PIK (peptide inhibitor of kinase), also demonstrated attenuated RILI. Taken together, these data suggests a key role for nmMLCK in vascular barrier regulation in RILI and warrants further examination of RILI strategies that target nmMLCK. PMID:27252850

  13. Olanzapine May Inhibit Colonic Motility Associated with the 5-HT Receptor and Myosin Light Chain Kinase

    PubMed Central

    Zhang, Jiarui; Qiao, Ying; Le, Jingjing

    2016-01-01

    Objective To study whether the effects of olanzapine on gastrointestinal motility is related to the serotonin antagonism and myosin light chain kinase. Methods Male Sprague-Dawley rats were randomly divided into four groups. Olanzapine gavage was performed for each treatment group during the course of 30 continuous days, while the same volume of saline was given to the rats in the control group. Defecation of the rats was observed on days 7 and 30 after olanzapine gavage. The effects of olanzapine on contraction of colonic smooth muscles were observed in ex vivo experiments. A Western blot was used to evaluate expression levels of the serotonin transporter (SERT) and MLCK in colon segments of the rats. Results ResultsaaCompared to the control group, 5-160 µ M of olanzapine could inhibit dose-dependently the contraction of colonic smooth muscle ex vivo experiments. The maximum smooth muscle contraction effects of 5-HT and acetylcholine significantly decreased after treatment with 40-160 µ M of olanzapine. Constipation was found in the olanzapine-treated rats on day 7 and have sustained day 30 after gavage. Expression of MLCK in olanzapine-treated rats was significantly decreased, whereas the expression of SERT significantly increased on the day 7, then significantly decreased on the day 30 after olanzapine gavage. Conclusion SERT and MLCK may involve in the inhibition of colonic contraction induced by olanzapine. PMID:27081386

  14. Mn2+ activates skinned smooth muscle cells in the absence of myosin light chain phosphorylation.

    PubMed

    Hoar, P E; Kerrick, W G

    1988-08-01

    Two effects of Mn2+ on skinned fibers from chicken gizzard smooth muscle were observed, dependent on the presence or absence of dithiothreitol (DTT) reducing agent. One involves protein oxidation (in the absence of DTT) with production of a "latch"-like state, and the other involves direct Mn2+ activation of contractile proteins. Cells activated by Mn2+ in the presence of ATP and the absence of Ca2+, Mg2+ and DTT did not relax when transferred to normal relaxing solutions. In contrast, when 5 mM DTT was included in the Mn2+ contracting solution to prevent protein oxidation by Mn2+, the cells still contracted when exposed to Mn2+, but relaxed rapidly when the Mn2+ was removed. In the presence of DTT both the Mn2+ activation and the relaxation following removal of Mn2+ were more rapid than normal Ca2+-activated contractions and relaxations. The skinned fibers activated by Mn2+ in the absence of DTT showed little active shortening unless DTT was added. This rigor-like state is probably due to oxidation of contractile proteins since the cells relaxed when exposed to a relaxing solution containing DTT (50 mM) and then contracted again in response to Ca2+ and relaxed normally. The Mn2+ activation was not associated with myosin light chain phosphorylation, in contrast to Ca2+-activated contractions. PMID:3186428

  15. Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury.

    PubMed

    Wang, Ting; Mathew, Biji; Wu, Xiaomin; Shimizu, Yuka; Rizzo, Alicia N; Dudek, Steven M; Weichselbaum, Ralph R; Jacobson, Jeffrey R; Hecker, Louise; Garcia, Joe G N

    2016-06-01

    Radiotherapy as a primary treatment for thoracic malignancies induces deleterious effects, such as acute or subacute radiation-induced lung injury (RILI). Although the molecular etiology of RILI is controversial and likely multifactorial, a potentially important cellular target is the lung endothelial cytoskeleton that regulates paracellular gap formation and the influx of macromolecules and fluid to the alveolar space. Here we investigate the central role of a key endothelial cytoskeletal regulatory protein, the nonmuscle isoform of myosin light chain kinase (nmMLCK), in an established murine RILI model. Our results indicate that thoracic irradiation significantly augmented nmMLCK protein expression and enzymatic activity in murine lungs. Furthermore, genetically engineered mice harboring a deletion of the nmMLCK gene (nmMLCK(-/-) mice) exhibited protection from RILI, as assessed by attenuated vascular leakage and leukocyte infiltration. In addition, irradiated wild-type mice treated with two distinct MLCK enzymatic inhibitors, ML-7 and PIK (peptide inhibitor of kinase), also demonstrated attenuated RILI. Taken together, these data suggests a key role for nmMLCK in vascular barrier regulation in RILI and warrants further examination of RILI strategies that target nmMLCK. PMID:27252850

  16. Canine cardiac myosin with special referrence to pressure overload cardiac hypertrophy. I. Subunit composition.

    PubMed

    Siemankowski, R F; Dreizen, P

    1978-12-10

    In studies of myosin from left and right ventricles of normal hearts and hypertrophic hearts at 5 weeks and 13 weeks after aortic banding, polyacrylamide gel electrophoresis shows intermediate molecular weight components which derive from heavy chains fragmented in the presence of dodecyl sulfate. The proportion of degraded heavy chains is greater in myosin from hypertrophic hearts than normal hearts, with comparable degradation in left and right ventricle myosin. The observed fragmentation of myosin results from proteolysis due to contaminant proteases or a thermally activated, heat-stable nonenzymatic process, or both. The susceptibility of heavy chains to crude myofibrillar proteases differs in normal and hypertrophic cardiac myosin; however, the kinetics of tryptic digestion are identical for both myosins. With precautions to minimize proteolytic artifacts on dodecyl sulfate-polyacrylamide gel electrophoresis, preparations of myosin from left and right ventricles of normal and hypertrophic hearts exhibit comparable subunit composition, with approximately molar ratios of heavy chains, light chain L1, and light chain L2. Comparable stoichiometry for the light chain fraction is determined by high speed sedimentation equilibrium at pH 11 and direct fractionation of the different cardiac myosins. We do not confirm reports (e.g. Wikman-Coffelt, J., Fenner, C., Smith, A., and Mason, D. T. (1975) J. Biol. Chem. 250, 1257-1262) of different proportions of light chains in left and right ventricle myosin of normal and hypertrophic canine hearts. The light chains display microheterogeneity, with L1 generating two isoelectric variants and L2 generating two major and two minor variants, but identical mobilities and isoelectric values are obtained in the different myosin preparations. PMID:152317

  17. Isolation of cardiac myosin light-chain isotypes by chromatofocusing. Comparison of human cardiac atrial light-chain 1 and foetal ventricular light-chain 1.

    PubMed

    Vincent, N D; Cummins, P

    1985-04-01

    Cardiac myosin light chain isotypes have been resolved using chromatofocusing, a new preparative column chromatographic technique. The method relies on production of narrow-range, shallow and stable pH gradients using ion-exchange resins and buffers with even buffering capacity over the required pH range. Light chains were resolved in order of decreasing isoelectric point in the pH range 5.2-4.5. Gradients of delta pH = 0.004-0.006/ml elution volume were achieved which were capable of resolving light chains with isoelectric point differences of only 0.03. Analytical isoelectric focusing of light chains in polyacrylamide gels could be used to predict the results of preparative chromatofocusing for method development. Chromatofocusing was capable of resolving human and bovine cardiac light chain 1 and 2 subunits, atrial (ALC) and ventricular (VLC) light chain isotypes and homologous VLC-2 and VLC-2* light chains. The technique was used to purify and resolve the human foetal ventricular light chain 1 (FLC-1) from adult ventricular light chain 1 (VLC-1) present in foetal ventricles and the atrial light chain 1 (ALC-1) in adult atria. Comparative peptide mapping studies and amino acid analyses were carried out on FLC-1 and ALC-1. No differences were detected between FLC-1 and ALC-1 using three different proteases and amino acid compositions were similar with the exception of glycine content. The studies indicate that FLC-1 and ALC-1 are homologous, and possibly identical, light chains. Comparison of human FLC-1/ALC-1 with VLC-1 suggested marked structural and chemical differences in these light chain isotypes, in particular in the contents of methionine, proline, lysine and alanine residues. Differences in the contents of these residues were also apparent in the corresponding bovine atrial and ventricular light chains [Wikman-Coffelt, J. & Srivastava, S. (1979) FEBS Lett. 106, 207-212]. The latter three residues are known to be rich in the N-termini of cardiac and

  18. Adiabatic compressibility of myosin subfragment-1 and heavy meromyosin with or without nucleotide.

    PubMed Central

    Tamura, Y; Suzuki, N; Mihashi, K

    1993-01-01

    The partial specific adiabatic compressibilities of myosin subfragment-1 (S1) and heavy meromyosin (HMM) of skeletal muscle in solution were determined by measuring the density and the sound velocity of the solution. The partial specific volumes of S1 and HMM were 0.713 and 0.711 cm3/g, respectively. The partial specific adiabatic compressibilities of S1 and HMM were 4.2 x 10(-12) and 2.9 x 10(-12) cm2/dyn, respectively. These values are in the same range as the most of globular proteins so far studied. The result indicates that the flexibility of S1 region almost equals to that of HMM. After binding to ADP.orthovanadate, S1 and HMM became softer than their complexes with ADP. The bulk moduli of S1 and HMM were of the order of (4-6) x 10(10) dyn/cm2, which are very comparable with the bulk modulus of muscle fiber. PMID:8298019

  19. Altered kinetics of contraction in skeletal muscle fibers containing a mutant myosin regulatory light chain with reduced divalent cation binding.

    PubMed Central

    Diffee, G M; Patel, J R; Reinach, F C; Greaser, M L; Moss, R L

    1996-01-01

    We examined the kinetic properties of rabbit skinned skeletal muscle fibers in which the endogenous myosin regulatory light chain (RLC) was partially replaced with a mutant RLC (D47A) containing a point mutation within the Ca2+/Mg2+ binding site that severely reduced its affinity for divalent cations. We found that when approximately 50% of the endogenous RLC was replaced by the mutant, maximum tension declined to approximately 60% of control and the rate constant of active tension redevelopment (ktr) after mechanical disruption of cross-bridges was reduced to approximately 70% of control. This reduction in ktr was not an indirect effect on kinetics due to a reduced number of strongly bound myosin heads, because when the strongly binding cross-bridge analog N-ethylmaleimide-modified myosin subfragment1 (NEM-S1) was added to the fibers, there was no effect upon maximum ktr. Fiber stiffness declined after D47A exchange in a manner indicative of a decrease in the number of strongly bound cross-bridges, suggesting that the force per cross-bridge was not significantly affected by the presence of D47A RLC. In contrast to the effects on ktr, the rate of tension relaxation in steadily activated fibers after flash photolysis of the Ca2+ chelator diazo-2 increased by nearly twofold after D47A exchange. We conclude that the incorporation of the nondivalent cation-binding mutant of myosin RLC decreases the proportion of cycling cross-bridges in a force-generating state by decreasing the rate of formation of force-generating bridges and increasing the rate of detachment. These results suggest that divalent cation binding to myosin RLC plays an important role in modulating the kinetics of cross-bridge attachment and detachment. Images FIGURE 2 PMID:8804617

  20. Hypertrophic cardiomyopathy associated Lys104Glu mutation in the myosin regulatory light chain causes diastolic disturbance in mice.

    PubMed

    Huang, Wenrui; Liang, Jingsheng; Kazmierczak, Katarzyna; Muthu, Priya; Duggal, Divya; Farman, Gerrie P; Sorensen, Lars; Pozios, Iraklis; Abraham, Theodore P; Moore, Jeffrey R; Borejdo, Julian; Szczesna-Cordary, Danuta

    2014-09-01

    We have examined, for the first time, the effects of the familial hypertrophic cardiomyopathy (HCM)-associated Lys104Glu mutation in the myosin regulatory light chain (RLC). Transgenic mice expressing the Lys104Glu substitution (Tg-MUT) were generated and the results were compared to Tg-WT (wild-type human ventricular RLC) mice. Echocardiography with pulse wave Doppler in 6month-old Tg-MUT showed early signs of diastolic disturbance with significantly reduced E/A transmitral velocities ratio. Invasive hemodynamics in 6month-old Tg-MUT mice also demonstrated a borderline significant prolonged isovolumic relaxation time (Tau) and a tendency for slower rate of pressure decline, suggesting alterations in diastolic function in Tg-MUT. Six month-old mutant animals had no LV hypertrophy; however, at >13months they displayed significant hypertrophy and fibrosis. In skinned papillary muscles from 5 to 6month-old mice a mutation induced reduction in maximal tension and slower muscle relaxation rates were observed. Mutated cross-bridges showed increased rates of binding to the thin filaments and a faster rate of the power stroke. In addition, ~2-fold lower level of RLC phosphorylation was observed in the mutant compared to Tg-WT. In line with the higher mitochondrial content seen in Tg-MUT hearts, the MUT-myosin ATPase activity was significantly higher than WT-myosin, indicating increased energy consumption. In the in vitro motility assay, MUT-myosin produced higher actin sliding velocity under zero load, but the velocity drastically decreased with applied load in the MUT vs. WT myosin. Our results suggest that diastolic disturbance (impaired muscle relaxation, lower E/A) and inefficiency of energy use (reduced contractile force and faster ATP consumption) may underlie the Lys104Glu-mediated HCM phenotype. PMID:24992035

  1. Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

    PubMed Central

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm

    2016-01-01

    Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease. PMID:27162358

  2. Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments.

    PubMed

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm

    2016-05-24

    Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease. PMID:27162358

  3. An isoform of myosin XI is responsible for the translocation of endoplasmic reticulum in tobacco cultured BY-2 cells

    PubMed Central

    Yokota, Etsuo; Ueda, Shunpei; Tamura, Kentaro; Orii, Hidefumi; Uchi, Satoko; Sonobe, Seiji; Hara-Nishimura, Ikuko; Shimmen, Teruo

    2009-01-01

    The involvement of myosin XI in generating the motive force for cytoplasmic streaming in plant cells is becoming evident. For a comprehensive understanding of the physiological roles of myosin XI isoforms, it is necessary to elucidate the properties and functions of each isoform individually. In tobacco cultured BY-2 cells, two types of myosins, one composed of 175 kDa heavy chain (175 kDa myosin) and the other of 170 kDa heavy chain (170 kDa myosin), have been identified biochemically and immunocytochemically. From sequence analyses of cDNA clones encoding heavy chains of 175 kDa and 170 kDa myosin, both myosins have been classified as myosin XI. Immunocytochemical studies using a polyclonal antibody against purified 175 kDa myosin heavy chain showed that the 175 kDa myosin is distributed throughout the cytoplasm as fine dots in interphase BY-2 cells. During mitosis, some parts of 175 kDa myosin were found to accumulate in the pre-prophase band (PPB), spindle, the equatorial plane of a phragmoplast and on the circumference of daughter nuclei. In transgenic BY-2 cells, in which an endoplasmic reticulum (ER)-specific retention signal, HDEL, tagged with green fluorescent protein (GFP) was stably expressed, ER showed a similar behaviour to that of 175 kDa myosin. Furthermore, this myosin was co-fractionated with GFP–ER by sucrose density gradient centrifugation. From these findings, it was suggested that the 175 kDa myosin is a molecular motor responsible for translocating ER in BY-2 cells. PMID:19039101

  4. X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibers.

    PubMed

    Yamaguchi, Maki; Kimura, Masako; Li, Zhao-Bo; Ohno, Tetsuo; Takemori, Shigeru; Hoh, Joseph F Y; Yagi, Naoto

    2016-04-15

    The phosphorylation of the myosin regulatory light chain (RLC) is an important modulator of skeletal muscle performance and plays a key role in posttetanic potentiation and staircase potentiation of twitch contractions. The structural basis for these phenomena within the filament lattice has not been thoroughly investigated. Using a synchrotron radiation source at SPring8, we obtained X-ray diffraction patterns from skinned rabbit psoas muscle fibers before and after phosphorylation of myosin RLC in the presence of myosin light chain kinase, calmodulin, and calcium at a concentration below the threshold for tension development ([Ca(2+)] = 10(-6.8)M). After phosphorylation, the first myosin layer line slightly decreased in intensity at ∼0.05 nm(-1)along the equatorial axis, indicating a partial loss of the helical order of myosin heads along the thick filament. Concomitantly, the (1,1/1,0) intensity ratio of the equatorial reflections increased. These results provide a firm structural basis for the hypothesis that phosphorylation of myosin RLC caused the myosin heads to move away from the thick filaments towards the thin filaments, thereby enhancing the probability of interaction with actin. In contrast, 2,3-butanedione monoxime (BDM), known to inhibit contraction by impeding phosphate release from myosin, had exactly the opposite effects on meridional and equatorial reflections to those of phosphorylation. We hypothesize that these antagonistic effects are due to the acceleration of phosphate release from myosin by phosphorylation and its inhibition by BDM, the consequent shifts in crossbridge equilibria leading to opposite changes in abundance of the myosin-ADP-inorganic phosphate complex state associated with helical order of thick filaments. PMID:26911280

  5. Constraints on intron evolution in the gene encoding the myosin alkali light chain in Drosophila

    SciTech Connect

    Leicht, B.G.; Muse, S.V.; Hanczyc, M.

    1995-01-01

    Interspecific comparisons of intron sequences reveal conserved blocks of invariant nucleotides and several other departures from the strictly neutral model of molecular evolution. To distinguish the past action of evolutionary forces in introns known to have regulatory information, we examined nucleotide sequence variation at 991 sites in a random sample of 16 Drosophila melanogaster alleles of the gene encoding the myosin alkali light chain (Mlc1). The Mlc1 gene of D. melanogaster encodes two Mlc1 isoforms via developmentally regulated alternative pre-mRNA splicing. Analyses of these data reveal that introns 4 and 5, which flank the alternatively spliced exon 5, have reduced levels of both intraspecific polymorphism and interspecific divergence relative to intron 3. No polymorphism was observed in any of the exons examined in D. melanogaster. A genealogical analysis clearly demonstrates the occurrence of intragenic recombination in the ancestral history of Mlc1. Recombination events are estimated to be 13 times more likely than mutation events over the span of the sequenced region. Although there is little evidence for pairwise linkage disequilibrium in the Mlc1 region, higher order disequilibrium. does seem to be present in the 5{prime} half of the portion of the gene that was examined. Predictions of the folding free energy of the pre-mRNA reveal that sampled alleles have a significantly higher (less stable) free energy than do randomly permuted sequences. These results are consistent with the hypothesis that introns surrounding an alternatively spliced exon are subjected to additional constraints, perhaps due to specific aspects of secondary structure required for appropriate splicing of the pre-mRNA molecule. 48 refs., 5 figs., 3 tabs.

  6. Myosin Light Chain Kinase Mediates Intestinal Barrier Disruption following Burn Injury

    PubMed Central

    Chen, Chuanli; Wang, Pei; Su, Qin; Wang, Shiliang; Wang, Fengjun

    2012-01-01

    Background Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC) phosphorylation mediated by MLC kinase (MLCK) is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction. Methodology/Principal Findings Male balb/c mice were assigned randomly to either sham burn (control) or 30% total body surface area (TBSA) full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg), an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC)-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression. Conclusions/Significance The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury. PMID:22529961

  7. [Usefulness of serum cardiac myosin light chain I for the estimation of acute myocardial infarction size].

    PubMed

    Narita, M; Kurihara, T; Murano, K; Usami, M

    1991-09-01

    To evaluate the usefulness of serum level of cardiac myosin light chain I (LC I) for the estimation of the extent of acute myocardial infarction (AMI), peak LC I level was compared with myocardial infarction weight (AMI weight) which was obtained by myocardial emission tomography with Tc-99m pyrophosphate (PYP). In 11 patients with AMI, serum LC I levels were measured once a day in most cases, and plasma CPK levels were measured serially (every 4 hours at least 48 hours after admission). Tc-99m PYP imagings were performed at second or third day of AMI, and AMI weight was calculated from the voxel numbers of myocardial hot spot in which Tc-99m PYP had accumulated. Peak LC I level correlated well with AMI weight (r = 0.72, p less than 0.02). As well as peak LC I level, peak CPK level correlated well with AMI weight (r = 0.68, p less than 0.05). But the estimation of the infarct size from peak LC I level had the following advantages over the estimation from peak CPK level. 1) We could compare peak LC I level with AMI weight in all 11 patients, but peak CPK level was able to compared with AMI weight in only 9 of them. This was because CPK level changed rapidly and reached maximum within 24 hours after the onset of AMI, while LC I level peaked after 3 to 5 days. 2) A good correlation between LC I and AMI weight was obtained by the determination of serum LC I level once a day.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1836269

  8. Effects of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin regulatory light chain.

    PubMed

    Espinoza-Fonseca, L Michel; Colson, Brett A; Thomas, David D

    2014-10-01

    We have performed 50 independent molecular dynamics (MD) simulations to determine the effect of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin (SMM) regulatory light chain (RLC). We previously showed that the N-terminal phosphorylation domain of RLC simultaneously populates two structural states in equilibrium, closed and open, and that phosphorylation at S19 induces a modest shift toward the open state, which is sufficient to activate smooth muscle. However, it remains unknown why pseudophosphorylation mutants poorly mimic phosphorylation-induced activation of SMM. We performed MD simulations of unphosphorylated, phosphorylated, and three pseudophosphorylated RLC mutants: S19E, T18D/S19D and T18E/S19E. We found that the S19E mutation does not shift the equilibrium toward the open state, indicating that simple charge replacement at position S19 does not mimic the activating effect of phosphorylation, providing a structural explanation for previously published functional data. In contrast, mutants T18D/S19D and T18E/S19E shift the equilibrium toward the open structure and partially activate in vitro motility, further supporting the model that an increase in the mol fraction of the open state is coupled to SMM motility. Structural analyses of the doubly-charged pseudophosphorylation mutants suggest that alterations in an interdomain salt bridge between residues R4 and D100 results in impaired signal transmission from RLC to the catalytic domain of SMM, which explains the low ATPase activity of these mutants. Our results demonstrate that phosphorylation produces a unique structural balance in the RLC. These observations have important implications for our understanding of the structural aspects of activation and force potentiation in smooth and striated muscle. PMID:25091814

  9. Regulation of myosin light chain phosphorylation in the trabecular meshwork: role in aqueous humour outflow facility.

    PubMed

    Rao, P Vasantha; Deng, Peifeng; Sasaki, Yasuharu; Epstein, David L

    2005-02-01

    Cellular contraction and relaxation and integrity of the actin cytoskeleton in trabecular meshwork (TM) tissue have been thought to influence aqueous humour outflow. However, the cellular pathways that regulate these events in TM cells are not well understood. In this study, we investigated physiological agonist-mediated regulation of myosin light chain (MLC) phosphorylation in the TM, and correlated such effects with alterations in aqueous outflow facility, since MLC phosphorylation is a critical biochemical determinant of cellular contraction in TM cells. Treatment of serum starved human TM cells with endothelin-1 (0.1 microM), thromboxane A2 mimetic U-46619 (1.0 microM), or angiotensin II (1 microM), all of which are agonists of G-protein coupled receptors, triggered activation of MLC phosphorylation, as determined by urea/glycerol-based Western blot analysis. Agonist-stimulated increase in MLC phosphorylation was associated with activation of Rho GTPase in TM cells, as determined in pull-down assays. In contrast, treatment of human TM cells with a novel Rho-kinase inhibitor H-1152 (0.1-2 microM), in the presence of serum reduced basal MLC phosphorylation. H-1152 also increased aqueous outflow facility significantly in a dose-dependent fashion, in perfusion studies with cadaver porcine eyes. This effect of H-1152 on outflow facility was associated with decreased MLC phosphorylation in TM tissue of drug-perfused eyes. Collectively, this study identifies potential physiological regulators of MLC phosphorylation in human TM cells and demonstrates the significance of Rho/Rho-kinase pathway-mediated MLC phosphorylation in modulation of aqueous outflow facility through TM. PMID:15670798

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

    SciTech Connect

    Muthu, Priya; Wang, Li; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Huang, Wenrui; Hernandez, Olga M.; Kawai, Masataka; Irving, Thomas C.; Szczesna-Cordary, Danuta

    2012-04-02

    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-{Delta}43 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 ({approx} 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{sub 1,1}/I{sub 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-{Delta}43. The equilibrium constant for the cross-bridge force generation step was smallest in Tg-{Delta}43. 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.

  11. Myosin light chain phosphorylation in contraction of gastric antral smooth muscle from neonate and adult rabbits.

    PubMed

    Ierardi, J A; Paul, D A; Ryan, J P

    1996-01-01

    The decreased contractility of gastric antral smooth muscle in the neonate has been attributed to reduced levels of activator calcium. It is generally accepted that calcium-dependent myosin light chain phosphorylation (MLCP) is the key step in the initiation of force development in smooth muscle. In this study, we investigated the relationship between MLCP and force development in gastric antral smooth muscle from neonatal (4-6 d old) and adult rabbits. We tested the hypothesis that the reduced force development of circular smooth muscle from the neonate would be accompanied by decreased levels of MLCP, as compared with data from adult animals. Full thickness muscle strips oriented parallel to the circular muscle layer were examined for their contractile response to acetylcholine (ACh) (10(-8) M to 10(-3) M) or 10(-4) M ACh only. In the latter study, tissues were rapidly frozen in a dry ice-acetone slurry for subsequent MLCP determination. MLCP was determined at times corresponding to 5, 10, 15, 30, and 60 s of stimulation. For each age group, maximal active force developed at an ACh concentration of 10(-4) M and was significantly greater in tissues from adults (1.86 +/- 0.24 N/m2, adult; 0.95 +/- 0.05 N/m2, neonate; p < 0.05). In contrast, no significant differences were observed with respect to basal or agonist-stimulated levels of MLCP. The data suggest that factors other than levels of MLCP contribute to the reduced force-generating capacity of antral smooth muscle from the neonate. PMID:8825402

  12. Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes.

    PubMed

    Dewerchin, Hannah L; Desmarets, Lowiese M; Noppe, Ytse; Nauwynck, Hans J

    2014-01-01

    Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV. PMID:24517254

  13. Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain.

    PubMed

    Guhathakurta, Piyali; Prochniewicz, Ewa; Thomas, David D

    2015-04-14

    We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40-45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC's location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders. PMID:25825773

  14. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

    PubMed Central

    Lima, V.V.; Lobato, N.S.; Filgueira, F.P.; Webb, R.C.; Tostes, R.C.; Giachini, F.R.

    2014-01-01

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction. PMID:25140811

  15. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain.

    PubMed

    Lima, V V; Lobato, N S; Filgueira, F P; Webb, R C; Tostes, R C; Giachini, F R

    2014-10-01

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction. PMID:25140811

  16. The force dependence of isometric and concentric potentiation in mouse muscle with and without skeletal myosin light chain kinase.

    PubMed

    Gittings, William; Aggarwal, Harish; Stull, James T; Vandenboom, Rene

    2015-01-01

    The isometric potentiation associated with myosin phosphorylation is force dependent. The purpose of this study was to assess the influence of a pre-existing period of isometric force on the concentric force potentiation displayed by mouse muscles with and without the ability to phosphorylate myosin. We tested isometric (ISO) and concentric (CON) potentiation, as well as concentric potentiation after isometric force (ISO-CON), in muscles from wild-type (WT) and skeletal myosin light chain kinase-deficient (skMLCK(-/-)) mice. A conditioning stimulus increased (i.e., potentiated) mean concentric force in the ISO-CON and CON conditions to 1.31 ± 0.02 and 1.35 ± 0.02 (WT) and to 1.19 ± 0.02 and 1.21 ± 0.01 (skMLCK(-/-)) of prestimulus levels, respectively (data n = 6-8, p < 0.05). No potentiation of mean isometric force was observed in either genotype. The potentiation of mean concentric force was inversely related to relative tetanic force level (P/Po) in both genotypes. Moreover, concentric potentiation varied greatly within each contraction type and was negatively correlated with unpotentiated force in both genotypes. Thus, although no effect of pre-existing force was observed, strong and inverse relationships between concentric force potentiation and unpotentiated concentric force may suggest an influence of attached and force-generating crossbridges on potentiation magnitude in both WT and skMLCK(-/-) muscles. PMID:25412230

  17. Tarantula myosin free head regulatory light chain phosphorylation stiffens N-terminal extension, releasing it and blocking its docking back.

    PubMed

    Alamo, Lorenzo; Li, Xiaochuan Edward; Espinoza-Fonseca, L Michel; Pinto, Antonio; Thomas, David D; Lehman, William; Padrón, Raúl

    2015-08-01

    Molecular dynamics simulations of smooth and striated muscle myosin regulatory light chain (RLC) N-terminal extension (NTE) showed that diphosphorylation induces a disorder-to-order transition. Our goal here was to further explore the effects of mono- and diphosphorylation on the straightening and rigidification of the tarantula myosin RLC NTE. For that we used MD simulations followed by persistence length analysis to explore the consequences of secondary and tertiary structure changes occurring on RLC NTE following phosphorylation. Static and dynamic persistence length analysis of tarantula RLC NTE peptides suggest that diphosphorylation produces an important 24-fold straightening and a 16-fold rigidification of the RLC NTE, while monophosphorylation has a less profound effect. This new information on myosin structural mechanics, not fully revealed by previous EM and MD studies, add support to a cooperative phosphorylation-dependent activation mechanism as proposed for the tarantula thick filament. Our results suggest that the RLC NTE straightening and rigidification after Ser45 phosphorylation leads to a release of the constitutively Ser35 monophosphorylated free head swaying away from the thick filament shaft. This is so because the stiffened diphosphorylated RLC NTE would hinder the docking back of the free head after swaying away, becoming released and mobile and unable to recover its original interacting position on activation. PMID:26038302

  18. New Isoform of Cardiac Myosin Light Chain Kinase and the Role of Cardiac Myosin Phosphorylation in α1-Adrenoceptor Mediated Inotropic Response

    PubMed Central

    Taniguchi, Masaya; Okamoto, Ryuji; Ito, Masaaki; Goto, Itaru; Fujita, Satoshi; Konishi, Katsuhisa; Mizutani, Hideo; Dohi, Kaoru; Hartshorne, David J.; Itoh, Takeo

    2015-01-01

    Background & Aims Cardiac myosin light chain kinase (cMLCK) plays an obligatory role in maintaining the phosphorylation levels of regulatory myosin light chain (MLC2), which is thought to be crucial for regulation of cardiac function. To test this hypothesis, the role played by ventricular MLC2 (MLC2v) phosphorylation was investigated in the phenylephrine-induced increase in twitch tension using the naturally-occurring mouse strain, C57BL/6N, in which cMLCK is down regulated. Methods and Results By Western blot and nanoLC-MS/MS analysis, cMLCKs with molecular mass of 61-kDa (cMLCK-2) and/or 86-kDa were identified in mice heart. Among various mouse strains, C57BL/6N expressed cMLCK-2 alone and the closest relative strain C57BL/6J expressed both cMLCKs. The levels of MLC2v phosphorylation was significantly lower in C57BL/6N than in C57BL/6J. The papillary muscle twitch tension induced by electrical field stimulation was smaller in C57BL/6N than C57BL/6J. Phenylephrine had no effect on MLC2v phosphorylation in either strains but increased the twitch tension more potently in C57BL/6J than in C57BL/6N. Calyculin A increased papillary muscle MLC2v phosphorylation to a similar extent in both strains but increased the phenylephrine-induced inotropic response only in C57BL/6N. There was a significant positive correlation between the phenylephrine-induced inotropic response and the levels of MLC2v phosphorylation within ranges of 15–30%. Conclusions We identified a new isoform of cMLCK with a molecular mass of 61kDa(cMLCK-2) in mouse heart. In the C57BL/6N strain, only cMLCK-2 was expressed and the basal MLC2v phosphorylation levels and the phenylephrine-induced inotropic response were both smaller. We suggest that a lower phenylephrine-induced inotropic response may be caused by the lower basal MLC2v phosphorylation levels in this strain. PMID:26512720

  19. Myosin regulatory light chain modulates the Ca2+ dependence of the kinetics of tension development in skeletal muscle fibers.

    PubMed Central

    Patel, J R; Diffee, G M; Moss, R L

    1996-01-01

    To determine the role of myosin regulatory light chain (RLC) in modulating contraction in skeletal muscle, we examined the rate of tension development in bundles of skinned skeletal muscle fibers as a function of the level of Ca(2+) activation after UV flash-induced release of Ca(2+) from the photosensitive Ca(2+) chelator DM-nitrophen. In control fiber bundles, the rate of tension development was highly dependent on the concentration of activator Ca(2+) after the flash. There was a greater than twofold increase in the rate of tension development when the post-flash [Ca(2+)] was increased from the lowest level tested (which produced a steady tension that was 42% of maximum tension) to the highest level (producing 97% of maximum tension). However, when 40-70% of endogenous myosin RLC was extracted from the fiber bundles, tension developed at the maximum rate, regardless of the post-flash concentration of Ca(2+). Thus, the Ca(2+) dependence of the rate of tension development was eliminated by partial extraction of myosin RLC, an effect that was partially reversed by recombination of RLC back into the fiber bundles. The elimination of the Ca(2+) dependence of the kinetics of tension development was specific to the extraction of RLC rather than an artifact of the co-extraction of both RLC and Troponin C, because the rate of tension development was still Ca(2+) dependent, even when nearly 50% of endogenous Troponin C was extracted from fiber bundles fully replete with RLC. Thus, myosin RLC appears to be a key component in modulating Ca(2+) sensitive cross-bridge transitions that limit the rate of force development after photorelease of Ca(2+) in skeletal muscle fibers. Images FIGURE 1 FIGURE 5 PMID:9172757

  20. Enhanced paracellular transport of insulin can be achieved via transient induction of myosin light chain phosphorylation

    PubMed Central

    Taverner, Alistair; Dondi, Ruggero; Almansour, Khaled; Laurent, Floriane; Owens, Siân-Eleri; Eggleston, Ian M.; Fotaki, Nikoletta; Mrsny, Randall J.

    2015-01-01

    The intestinal epithelium functions to effectively restrict the causal uptake of luminal contents but has been demonstrated to transiently increase paracellular permeability properties to provide an additional entry route for dietary macromolecules. We have examined a method to emulate this endogenous mechanism as a means of enhancing the oral uptake of insulin. Two sets of stable Permeant Inhibitor of Phosphatase (PIP) peptides were rationally designed to stimulate phosphorylation of intracellular epithelial myosin light chain (MLC) and screened using Caco-2 monolayers in vitro. Apical application of PIP peptide 640, designed to disrupt protein–protein interactions between protein phosphatase 1 (PP1) and its regulator CPI-17, resulted in a reversible and non-toxic transient reduction in Caco-2 monolayer trans-epithelial electric resistance (TEER) and opening of the paracellular route to 4 kDa fluorescent dextran but not 70 kDa dextran in vitro. Apical application of PIP peptide 250, designed to impede MYPT1-mediated regulation of PP1, also decreased TEER in a reversible and non-toxic manner but transiently opened the paracellular route to both 4 and 70 kDa fluorescent dextrans. Direct injection of PIP peptides 640 or 250 with human insulin into the lumen of rat jejunum caused a decrease in blood glucose levels that was PIP peptide and insulin dose-dependent and correlated with increased pMLC levels. Systemic levels of insulin suggested approximately 3–4% of the dose injected into the intestinal lumen was absorbed, relative to a subcutaneous injection. Measurement of insulin levels in the portal vein showed a time window of absorption that was consistent with systemic concentration-time profiles and approximately 50% first-pass clearance by the liver. Monitoring the uptake of a fluorescent form of insulin suggested its uptake occurred via the paracellular route. Together, these studies add validation to the presence of an endogenous mechanism used by the

  1. Mutations of the Drosophila Myosin Regulatory Light Chain Affect Courtship Song and Reduce Reproductive Success

    PubMed Central

    Chakravorty, Samya; Vu, Hien; Foelber, Veronica; Vigoreaux, Jim O.

    2014-01-01

    The Drosophila indirect flight muscles (IFM) rely on an enhanced stretch-activation response to generate high power output for flight. The IFM is neurally activated during the male courtship song, but its role, if any, in generating the small amplitude wing vibrations that produce the song is not known. Here, we examined the courtship song properties and mating behavior of three mutant strains of the myosin regulatory light chain (DMLC2) that are known to affect IFM contractile properties and impair flight: (i) Dmlc2Δ2–46 (Ext), an N-terminal extension truncation; (ii) Dmlc2S66A,S67A (Phos), a disruption of two MLC kinase phosphorylation sites; and (iii) Dmlc2Δ2–46;S66A,S67A (Dual), expressing both mutations. Our results show that the Dmlc2 gene is pleiotropic and that mutations that have a profound effect on flight mechanics (Phos and Dual) have minimal effects on courtship song. None of the mutations affect interpulse interval (IPI), a determinant of species-specific song, and intrapulse frequency (IPF) compared to Control (Dmlc2+ rescued null strain). However, abnormalities in the sine song (increased frequency) and the pulse song (increased cycles per pulse and pulse length) evident in Ext males are not apparent in Dual males suggesting that Ext and Phos interact differently in song and flight mechanics, given their known additive effect on the latter. All three mutant males produce a less vigorous pulse song and exhibit impaired mating behavior compared to Control males. As a result, females are less receptive to Ext, Phos, and Dual males when a Control male is present. These results open the possibility that DMLC2, and perhaps contractile protein genes in general, are partly under sexual selection. That mutations in DMLC2 manifest differently in song and flight suggest that this protein fulfills different roles in song and flight and that stretch activation plays a smaller role in song production than in flight. PMID:24587213

  2. Definite differences between in vitro actin-myosin sliding and muscle contraction as revealed using antibodies to myosin head.

    PubMed

    Sugi, Haruo; Chaen, Shigeru; Kobayashi, Takakazu; Abe, Takahiro; Kimura, Kazushige; Saeki, Yasutake; Ohnuki, Yoshiki; Miyakawa, Takuya; Tanokura, Masaru; Sugiura, Seiryo

    2014-01-01

    Muscle contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated contraction of muscle fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor muscle fiber contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on muscle contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in muscle fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and muscle contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly

  3. IQ motif selectivity in human IQGAP1: binding of myosin essential light chain and S100B.

    PubMed

    Pathmanathan, Sevvel; Elliott, Sarah F; McSwiggen, Sara; Greer, Brett; Harriott, Pat; Irvine, G Brent; Timson, David J

    2008-11-01

    IQGAPs are cytoskeletal scaffolding proteins which link signalling pathways to the reorganisation of actin and microtubules. Human IQGAP1 has four IQ motifs each of which binds to calmodulin. The same region has been implicated in binding to two calmodulin-like proteins, the myosin essential light chain Mlc1sa and the calcium and zinc ion binding protein S100B. Using synthetic peptides corresponding to the four IQ motifs of human IQGAP1, we showed by native gel electrophoresis that only the first IQ motif interacts with Mlc1sa. This IQ motif, and also the fourth, interacts with the budding yeast myosin essential light chain Mlc1p. The first and second IQ motifs interact with S100B in the presence of calcium ions. This clearly establishes that S100B can interact with its targets through IQ motifs in addition to interacting via previously reported sequences. These results are discussed in terms of the function of IQGAP1 and IQ motif recognition. PMID:18587628

  4. TNF causes changes in glomerular endothelial permeability and morphology through a Rho and myosin light chain kinase-dependent mechanism.

    PubMed

    Xu, Chang; Wu, Xiaoyan; Hack, Bradley K; Bao, Lihua; Cunningham, Patrick N

    2015-12-01

    A key function of the endothelium is to serve as a regulated barrier between tissue compartments. We have previously shown that tumor necrosis factor (TNF) plays a crucial role in lipopolysaccharide (LPS)-induced acute kidney injury, in part by causing injury to the renal endothelium through its receptor TNFR1. Here, we report that TNF increased permeability to albumin in primary culture mouse renal endothelial cells, as well as human glomerular endothelial cells. This process occurred in association with changes in the actin cytoskeleton and was associated with gaps between previously confluent cells in culture and decreases in the tight junction protein occludin. This process was dependent on myosin light chain activation, as seen by its prevention with Rho-associated kinase and myosin light chain kinase (MLCK) inhibitors. Surprisingly, permeability was not blocked by inhibition of apoptosis with caspase inhibitors. Additionally, we found that the renal glycocalyx, which plays an important role in barrier function, was also degraded by TNF in a Rho and MLCK dependent fashion. TNF treatment caused a decrease in the size of endothelial fenestrae, dependent on Rho and MLCK, although the relevance of this to changes in permeability is uncertain. In summary, TNF-induced barrier dysfunction in renal endothelial cells is crucially dependent upon the Rho/MLCK signaling pathway. PMID:26634902

  5. Pre-steady-state kinetics of the activation of rabbit skeletal muscle myosin light chain kinase by Ca2+/calmodulin.

    PubMed

    Bowman, B F; Peterson, J A; Stull, J T

    1992-03-15

    Myosin light chain kinase is activated by Ca2+/calmodulin. Insights into the kinetic mechanism of this activation by Ca2+/calmodulin have now been obtained using extrinsically labeled fluorescent calmodulin, a fluorescent peptide substrate, and a stopped-flow spectrophotofluorimeter. We employed spinach calmodulin labeled with the sulfhydryl-selective probe, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid, to measure changes in the fluorescence intensity of the 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin upon binding to rabbit skeletal muscle myosin light chain kinase. The fluorescent peptide substrate KKRAARAC(sulfobenzo-furazan)SNVFS-amide was used to measure kinase activity. Our results showed that the binding interaction could be modeled as a two-step process: a bimolecular reaction with an association rate of 4.6 x 10(7) M-1 s-1 followed by an isomerization with a rate of 2.2 s-1. Phosphorylation of the peptide during stopped-flow experiments could be modeled by a two-step process with a catalytic association rate of 6.5 x 10(6) M-1 s-1 and a turnover rate of 10-20 s-1. Our results also indicated that kinase activity occurred too rapidly for the slower isomerization rate of 2.2 s-1 to be linked specifically to the activation process. PMID:1544916

  6. Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

    SciTech Connect

    Colson, Brett A.; Locher, Matthew R.; Bekyarova, Tanya; Patel, Jitandrakumar R.; Fitzsimons, Daniel P.; Irving, Thomas C.; Moss, Richard L.

    2010-05-25

    Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca{sup 2+} sensitivity of force (pCa{sub 50}), PKA treatment has been shown to decrease pCa{sub 50}, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca{sup 2+}-independent force and maximum Ca{sup 2+}-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I{sub 1,1}/I{sub 1,0}) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d{sub 1,0}) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I{sub 1,1}/I{sub 1,0} and, as hypothesized, treatment with MLCK also increased I{sub 1,1}/I{sub 1,0}, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by {approx}2 nm ({Delta} 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca{sup 2+} sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

  7. Erythrocyte Protein 4.1 Binds and Regulates Myosin

    NASA Astrophysics Data System (ADS)

    Pasternack, Gary R.; Racusen, Richard H.

    1989-12-01

    Myosin was recently identified in erythrocytes and was shown to partition both with membrane and cytosolic fractions, suggesting that it may be loosely bound to membranes [Fowler, V. M., Davis, J. Q. & Bennett, V. (1985) J. Cell Biol. 100, 47-55, and Wong, A. J., Kiehart, D. P. & Pollard, T. D. (1985) J. Biol. Chem. 260, 46-49]; however, the molecular basis for this binding was unclear. The present studies employed immobilized monomeric myosin to examine the interaction of myosin with erythrocyte protein 4.1. In human erythrocytes, protein 4.1 binds to integral membrane proteins and mediates spectrin-actin assembly. Protein 4.1 binds to rabbit skeletal muscle myosin with a Kd = 140 nM and a stoichiometry consistent with 1:1 binding. Heavy meromyosin competes for protein 4.1 binding with Ki = 36-54 nM; however, the S1 fragment (the myosin head) competes less efficiently. Affinity chromatography of partial chymotryptic digests of protein 4.1 on immobilized myosin identified a 10-kDa domain of protein 4.1 as the myosin-binding site. In functional studies, protein 4.1 partially inhibited the actin-activated Mg2+-ATPase activity of rabbit skeletal muscle myosin with Ki = 51 nM. Liver cytosolic and erythrocyte myosins preactivated with myosin light-chain kinase were similarly inhibited by protein 4.1. These studies show that protein 4.1 binds, modulates, and thus may regulate myosin. This interaction might serve to generate the contractile forces involved in Mg2+-ATP-dependent shape changes in erythrocytes and may additionally serve as a model for myosin organization and regulation in non-muscle cells.

  8. The pepsin digestibility of thermal gel products made from white croaker (Pennahia argentata) muscle in associating with myosin polymerization levels.

    PubMed

    Ueki, Nobuhiko; Wan, Jianrong; Watabe, Shugo

    2014-12-01

    Thermal gels were made from white croaker (Pennahia argentata) surimi at various polymerization levels of myosin heavy chains induced by suwari treatment at 38 °C for various time periods and subsequently heated at 85 °C for 20 min. Myosin heavy chain polymerization levels were also achieved in the presence of microbial transglutaminase (MTG) added at various concentrations in the surimi. The breaking strength and breaking strain rate were markedly increased during suwari treatment up to 60 min in accordance with the increased levels of myosin heavy chain polymerization. MTG enhanced myosin heavy chain polymerization during suwari treatment for 15 and 30 min, resulting in the increase of breaking strength. The solubilization in 8 M urea and pepsin digestibility of these gels as well as angiotensin I-converting enzyme (ACE) inhibitory activity of their pepsin digests were decreased with the increased levels of myosin heavy chain polymerization. These results suggest that myosin heavy chain polymerization affects not only rheological properties of thermal gels but also their functional properties for human health. PMID:25399805

  9. The modulatory effect of MgATP on heterotrimeric smooth muscle myosin phosphatase activity.

    PubMed

    Sato, O; Ogawa, Y

    1999-10-01

    Regulation of the enzymatic activity of heterotrimeric smooth muscle myosin phosphatase (SMMP) by MgATP was examined using phosphorylated myosin (P-myosin), heavy meromyosin (P-HMM), subfragment-1 (P-S1), and 20 kDa myosin light chain (P-MLC(20)) as substrates. The activity toward P-myosin and P-HMM was dose-dependently reduced by MgATP, whereas that toward P-S1 or P-MLC(20) was unchanged. The reduction was mainly due to a decrease in the affinity of SMMP for the substrate with the unchanged maximum activity. This regulation is entirely new in the respect that the responsible molecule is the substrate, not SMMP. Because P-myosin derived from myosin stored in 50% glycerol at -20 degrees C was insensitive to MgATP, the proper integrity of P-myosin is required. Coexisting myosin did not affect this regulation, but it inhibited the SMMP activity in the absence of MgATP. With P-myosin, the enzyme activity was biphasically steeply dependent on the ionic strength. This requires that determinations are conducted with a fixed ionic strength. The Q(10) value was about 2, which was quite similar to that for myosin light chain kinase. These results suggest that the rate of dephosphorylation of P-myosin is lowered at rest, but that it may reach a value comparable to the rate of phosphorylation of myosin in the sarcoplasm with the increased level of P-myosin during muscle activation. This regulation by MgATP may underlie the "latch mechanism" in some respects. PMID:10502690

  10. The properties of the actin-myosin interaction in the heart muscle depend on the isoforms of myosin but not of α-actin.

    PubMed

    Kopylova, G; Nabiev, S; Nikitina, L; Shchepkin, D; Bershitsky, S

    2016-08-01

    In myocardium of mammals there are two isoforms of myosin heavy chains, α and β. In ventricle, together with ventricular isoforms of light chains they form two isomyosins: V1 and V3, homodimers of α- and β-heavy chains, respectively. In atria, α- and β-heavy chains together with atrial light chains form A1 (αα) and A2 (ββ) isomyosins. Besides in myocardium two isoforms of α-actin, skeletal and cardiac, are expressed. We assume that the differences in the amino acid sequence of cardiac and skeletal actin may affect its interaction with myosin. To test this hypothesis, we investigated characteristics of actin-myosin interactions of cardiac and skeletal isoforms of α-actin with the isoforms of cardiac myosin using an optical trap technique and an in vitro motility assay. It was found that the mechanical and kinetic characteristics of the interactions of the isoforms of cardiac myosin with actin depend on the isoforms of myosin not α-actin. PMID:27264951

  11. Neuregulin1-β decreases interleukin-1β-induced RhoA activation, myosin light chain phosphorylation, and endothelial hyperpermeability.

    PubMed

    Wu, Limin; Ramirez, Servio H; Andrews, Allison M; Leung, Wendy; Itoh, Kanako; Wu, Jiang; Arai, Ken; Lo, Eng H; Lok, Josephine

    2016-01-01

    Neuregulin-1 (NRG1) is an endogenous growth factor with multiple functions in the embryonic and postnatal brain. The NRG1 gene is large and complex, transcribing more than twenty transmembrane proteins and generating a large number of isoforms in tissue and cell type-specific patterns. Within the brain, NRG1 functions have been studied most extensively in neurons and glia, as well as in the peripheral vasculature. Recently, NRG1 signaling has been found to be important in the function of brain microvascular endothelial cells, decreasing IL-1β-induced increases in endothelial permeability. In the current experiments, we have investigated the pathways through which the NRG1-β isoform acts on IL-1β-induced endothelial permeability. Our data show that NRG1-β increases barrier function, measured by transendothelial electrical resistance, and decreases IL-1β-induced hyperpermeability, measured by dextran-40 extravasation through a monolayer of brain microvascular endothelial cells plated on transwells. An investigation of key signaling proteins suggests that the effect of NRG1-β on endothelial permeability is mediated through RhoA activation and myosin light chain phosphorylation, events which affect filamentous actin morphology. In addition, AG825, an inhibitor of the erbB2-associated tyrosine kinase, reduces the effect of NRG1-β on IL-1β-induced RhoA activation and myosin light chain phosphorylation. These data add to the evidence that NRG1-β signaling affects changes in the brain microvasculature in the setting of neuroinflammation. We propose the following events for neuregulin-1-mediated effects on Interleukin-1 β (IL-1β)-induced endothelial hyperpermeability: IL-1β leads to RhoA activation, resulting in an increase in phosphorylation of myosin light chain (MLC). Phosphorylation of MLC is known to result in actin contraction and alterations in the f-actin cytoskeletal structure. These changes are associated with increased endothelial permeability

  12. Regulation of calcium channels in smooth muscle: New insights into the role of myosin light chain kinase

    PubMed Central

    Martinsen, A; Dessy, C; Morel, N

    2014-01-01

    Smooth muscle myosin light chain kinase (MLCK) plays a crucial role in artery contraction, which regulates blood pressure and blood flow distribution. In addition to this role, MLCK contributes to Ca2+ flux regulation in vascular smooth muscle (VSM) and in non-muscle cells, where cytoskeleton has been suggested to help Ca2+ channels trafficking. This conclusion is based on the use of pharmacological inhibitors of MLCK and molecular and cellular techniques developed to down-regulate the enzyme. Dissimilarities have been observed between cells and whole tissues, as well as between large conductance and small resistance arteries. A differential expression in MLCK and ion channels (either voltage-dependent Ca2+ channels or non-selective cationic channels) could account for these observations, and is in line with the functional properties of the arteries. A potential involvement of MLCK in the pathways modulating Ca2+ entry in VSM is described in the present review. PMID:25483583

  13. IQ-motif selectivity in human IQGAP2 and IQGAP3: binding of calmodulin and myosin essential light chain.

    PubMed

    Atcheson, Erwan; Hamilton, Elaine; Pathmanathan, Sevvel; Greer, Brett; Harriott, Pat; Timson, David J

    2011-10-01

    The IQGAP [IQ-motif-containing GAP (GTPase-activating protein)] family members are eukaryotic proteins that act at the interface between cellular signalling and the cytoskeleton. As such they collect numerous inputs from a variety of signalling pathways. A key binding partner is the calcium-sensing protein CaM (calmodulin). This protein binds mainly through a series of IQ-motifs which are located towards the middle of the primary sequence of the IQGAPs. In some IQGAPs, these motifs also provide binding sites for CaM-like proteins such as myosin essential light chain and S100B. Using synthetic peptides and native gel electrophoresis, the binding properties of the IQ-motifs from human IQGAP2 and IQGAP3 have been mapped. The second and third IQ-motifs in IQGAP2 and all four of the IQ-motifs of IQGAP3 interacted with CaM in the presence of calcium ions. However, there were differences in the type of interaction: while some IQ-motifs were able to form complexes with CaM which were stable under the conditions of the experiment, others formed more transient interactions. The first IQ-motifs from IQGAP2 and IQGAP3 formed transient interactions with CaM in the absence of calcium and the first motif from IQGAP3 formed a transient interaction with the myosin essential light chain Mlc1sa. None of these IQ-motifs interacted with S100B. Molecular modelling suggested that all of the IQ-motifs, except the first one from IQGAP2 formed α-helices in solution. These results extend our knowledge of the selectivity of IQ-motifs for CaM and related proteins. PMID:21299499

  14. IQ-motif selectivity in human IQGAP2 and IQGAP3: binding of calmodulin and myosin essential light chain

    PubMed Central

    Atcheson, Erwan; Hamilton, Elaine; Pathmanathan, Sevvel; Greer, Brett; Harriott, Pat; Timson, David J.

    2011-01-01

    The IQGAP [IQ-motif-containing GAP (GTPase-activating protein)] family members are eukaryotic proteins that act at the interface between cellular signalling and the cytoskeleton. As such they collect numerous inputs from a variety of signalling pathways. A key binding partner is the calcium-sensing protein CaM (calmodulin). This protein binds mainly through a series of IQ-motifs which are located towards the middle of the primary sequence of the IQGAPs. In some IQGAPs, these motifs also provide binding sites for CaM-like proteins such as myosin essential light chain and S100B. Using synthetic peptides and native gel electrophoresis, the binding properties of the IQ-motifs from human IQGAP2 and IQGAP3 have been mapped. The second and third IQ-motifs in IQGAP2 and all four of the IQ-motifs of IQGAP3 interacted with CaM in the presence of calcium ions. However, there were differences in the type of interaction: while some IQ-motifs were able to form complexes with CaM which were stable under the conditions of the experiment, others formed more transient interactions. The first IQ-motifs from IQGAP2 and IQGAP3 formed transient interactions with CaM in the absence of calcium and the first motif from IQGAP3 formed a transient interaction with the myosin essential light chain Mlc1sa. None of these IQ-motifs interacted with S100B. Molecular modelling suggested that all of the IQ-motifs, except the first one from IQGAP2 formed α-helices in solution. These results extend our knowledge of the selectivity of IQ-motifs for CaM and related proteins. PMID:21299499

  15. Cardiac myosin light chain phosphorylation and inotropic effects of a biased ligand, TRV120023, in a dilated cardiomyopathy model

    PubMed Central

    Tarigopula, Madhusudhan; Davis, Robert T.; Mungai, Paul T.; Ryba, David M.; Wieczorek, David F.; Cowan, Conrad L.; Violin, Jonathan D.; Wolska, Beata M.; Solaro, R. John

    2015-01-01

    Aims Therapeutic approaches to treat familial dilated cardiomyopathy (DCM), which is characterized by depressed sarcomeric tension and susceptibility to Ca2+-related arrhythmias, have been generally unsuccessful. Our objective in the present work was to determine the effect of the angiotensin II type 1 receptor (AT1R) biased ligand, TRV120023, on contractility of hearts of a transgenic mouse model of familial DCM with mutation in tropomyosin at position 54 (TG-E54K). Our rationale is based on previous studies, which have supported the hypothesis that biased G-protein-coupled receptor ligands, signalling via β-arrestin, increase cardiac contractility with no effect on Ca2+ transients. Our previous work demonstrated that the biased ligand TRV120023 is able to block angiotensin-induced hypertrophy, while promoting an increase in sarcomere Ca2+ response. Methods and results We tested the hypothesis that the depression in cardiac function associated with DCM can be offset by infusion of the AT1R biased ligand, TRV120023. We intravenously infused saline, TRV120023, or the unbiased ligand, losartan, for 15 min in TG-E54K and non-transgenic mice to obtain left ventricular pressure–volume relations. Hearts were analysed for sarcomeric protein phosphorylation. Results showed that the AT1R biased ligand increases cardiac performance in TG-E54K mice in association with increased myosin light chain-2 phosphorylation. Conclusion Treatment of mice with an AT1R biased ligand, acting via β-arrestin signalling, is able to induce an increase in cardiac contractility associated with an increase in ventricular myosin light chain-2 phosphorylation. AT1R biased ligands may prove to be a novel inotropic approach in familial DCM. PMID:26045475

  16. Evolution of the Dynein Heavy Chain Family in Ciliates.

    PubMed

    Rajagopalan, Vidyalakshmi; Wilkes, David E

    2016-01-01

    Dynein heavy chains are motor proteins that comprise a large gene family found across eukaryotes. We have investigated this gene family in four ciliate species: Ichthyophthirius, Oxytricha, Paramecium, and Tetrahymena. Ciliates appear to encode more dynein heavy chain genes than most eukaryotes. Phylogenetic comparisons demonstrated that the last common ancestor of the ciliates that were examined expressed at least 14 types of dynein heavy chains with most of the expansion coming from the single-headed inner arm dyneins. Each of the dyneins most likely performed different functions within the cell. PMID:26084401

  17. Phosphorylation and the N-terminal extension of the regulatory light chain help orient and align the myosin heads in Drosophila flight muscle

    SciTech Connect

    Farman, Gerrie P.; Miller, Mark S.; Reedy, Mary C.; Soto-Adames, Felipe N.; Vigoreaux, Jim O.; Maughan, David W.; Irving, Thomas C.

    2010-02-02

    X-ray diffraction of the indirect flight muscle (IFM) in living Drosophila at rest and electron microscopy of intact and glycerinated IFM was used to compare the effects of mutations in the regulatory light chain (RLC) on sarcomeric structure. Truncation of the RLC N-terminal extension (Dmlc2{sup {Delta}2-46}) or disruption of the phosphorylation sites by substituting alanines (Dmlc2{sup S66A, S67A}) decreased the equatorial intensity ratio (I{sub 20}/I{sub 10}), indicating decreased myosin mass associated with the thin filaments. Phosphorylation site disruption (Dmlc2{sup S66A, S67A}), but not N-terminal extension truncation (Dmlc2{sup {Delta}2-46}), decreased the 14.5 nm reflection intensity, indicating a spread of the axial distribution of the myosin heads. The arrangement of thick filaments and myosin heads in electron micrographs of the phosphorylation mutant (Dmlc2{sup S66A, S67A}) appeared normal in the relaxed and rigor states, but when calcium activated, fewer myosin heads formed cross-bridges. In transgenic flies with both alterations to the RLC (Dmlc2{sup {Delta}2-46; S66A, S67A}), the effects of the dual mutation were additive. The results suggest that the RLC N-terminal extension serves as a 'tether' to help pre-position the myosin heads for attachment to actin, while phosphorylation of the RLC promotes head orientations that allow optimal interactions with the thin filament.

  18. Orthologous myosin isoforms and scaling of shortening velocity with body size in mouse, rat, rabbit and human muscles

    PubMed Central

    Pellegrino, M A; Canepari, M; Rossi, R; D'Antona, G; Reggiani, C; Bottinelli, R

    2003-01-01

    Maximum shortening velocity (V0) was determined in single fibres dissected from hind limb skeletal muscles of rabbit and mouse and classified according to their myosin heavy chain (MHC) isoform composition. The values for rabbit and mouse V0 were compared with the values previously obtained in man and rat under identical experimental conditions. Significant differences in V0 were found between fibres containing corresponding myosin isoforms in different species: as a general rule for each isoform V0 decreased with body mass. Myosin isoform distributions of soleus and tibialis anterior were analysed in mouse, rat, rabbit and man: the proportion of slow myosin generally increased with increasing body size. The diversity between V0 of corresponding myosin isoforms and the different myosin isoform composition of corresponding muscles determine the scaling of shortening velocity of whole muscles with body size, which is essential for optimisation of locomotion. The speed of actin translocation (Vf) in in vitro motility assay was determined with myosins extracted from single muscle fibres of all four species: significant differences were found between myosin isoforms in each species and between corresponding myosin isoforms in different species. The values of V0 and Vf determined for each myosin isoform were significantly correlated, strongly supporting the view that the myosin isoform expressed is the major determinant of maximum shortening velocity in muscle fibres. PMID:12562996

  19. Excessive Myosin Activity in Mbs Mutants Causes Photoreceptor Movement Out of the Drosophila Eye Disc Epithelium

    PubMed Central

    Lee, Arnold; Treisman, Jessica E.

    2004-01-01

    Neuronal cells must extend a motile growth cone while maintaining the cell body in its original position. In migrating cells, myosin contraction provides the driving force that pulls the rear of the cell toward the leading edge. We have characterized the function of myosin light chain phosphatase, which down-regulates myosin activity, in Drosophila photoreceptor neurons. Mutations in the gene encoding the myosin binding subunit of this enzyme cause photoreceptors to drop out of the eye disc epithelium and move toward and through the optic stalk. We show that this phenotype is due to excessive phosphorylation of the myosin regulatory light chain Spaghetti squash rather than another potential substrate, Moesin, and that it requires the nonmuscle myosin II heavy chain Zipper. Myosin binding subunit mutant cells continue to express apical epithelial markers and do not undergo ectopic apical constriction. In addition, mutant cells in the wing disc remain within the epithelium and differentiate abnormal wing hairs. We suggest that excessive myosin activity in photoreceptor neurons may pull the cell bodies toward the growth cones in a process resembling normal cell migration. PMID:15075368

  20. The neck region of the myosin motor domain acts as a lever arm to generate movement.

    PubMed Central

    Uyeda, T Q; Abramson, P D; Spudich, J A

    1996-01-01

    The myosin head consists of a globular catalytic domain that binds actin and hydrolyzes ATP and a neck domain that consists of essential and regulatory light chains bound to a long alpha-helical portion of the heavy chain. The swinging neck-level model assumes that a swinging motion of the neck relative to the catalytic domain is the origin of movement. This model predicts that the step size, and consequently the sliding velocity, are linearly related to the length of the neck. We have tested this point by characterizing a series of mutant Dictyostelium myosins that have different neck lengths. The 2xELCBS mutant has an extra binding site for essential light chain. The delta RLCBS mutant myosin has an internal deletion that removes the regulatory light chain binding site. The delta BLCBS mutant lacks both light chain binding sites. Wild-type myosin and these mutant myosins were subjected to the sliding filament in vitro motility assay. As expected, mutants with shorter necks move slower than wild-type myosin in vitro. Most significantly, a mutant with a longer neck moves faster than the wild type, and the sliding velocities of these myosins are linearly related to the neck length, as predicted by the swinging neck-lever model. A simple extrapolation to zero speed predicts that the fulcrum point is in the vicinity of the SH1-SH2 region in the catalytic domain. Images Fig. 1 Fig. 2 Fig. 3 PMID:8633089

  1. Two distinct myosin II populations coordinate ovulatory contraction of the myoepithelial sheath in the Caenorhabditis elegans somatic gonad

    PubMed Central

    Ono, Kanako; Ono, Shoichiro

    2016-01-01

    The myoepithelial sheath in the somatic gonad of the nematode Caenorhabditis elegans has nonstriated contractile actomyosin networks that produce highly coordinated contractility for ovulation of mature oocytes. Two myosin heavy chains are expressed in the myoepithelial sheath, which are also expressed in the body-wall striated muscle. The troponin/tropomyosin system is also present and essential for ovulation. Therefore, although the myoepithelial sheath has smooth muscle–like contractile apparatuses, it has a striated muscle–like regulatory mechanism through troponin/tropomyosin. Here we report that the myoepithelial sheath has a distinct myosin population containing nonmuscle myosin II isoforms, which is regulated by phosphorylation and essential for ovulation. MLC-4, a nonmuscle myosin regulatory light chain, localizes to small punctate structures and does not colocalize with large, needle-like myosin filaments containing MYO-3, a striated-muscle myosin isoform. RNA interference of MLC-4, as well as of its upstream regulators, LET-502 (Rho-associated coiled-coil forming kinase) and MEL-11 (a myosin-binding subunit of myosin phosphatase), impairs ovulation. Expression of a phosphomimetic MLC-4 mutant mimicking a constitutively active state also impairs ovulation. A striated-muscle myosin (UNC-54) appears to provide partially compensatory contractility. Thus the results indicate that the two spatially distinct myosin II populations coordinately regulate ovulatory contraction of the myoepithelial sheath. PMID:26864628

  2. Two distinct myosin II populations coordinate ovulatory contraction of the myoepithelial sheath in the Caenorhabditis elegans somatic gonad.

    PubMed

    Ono, Kanako; Ono, Shoichiro

    2016-04-01

    The myoepithelial sheath in the somatic gonad of the nematodeCaenorhabditis eleganshas nonstriated contractile actomyosin networks that produce highly coordinated contractility for ovulation of mature oocytes. Two myosin heavy chains are expressed in the myoepithelial sheath, which are also expressed in the body-wall striated muscle. The troponin/tropomyosin system is also present and essential for ovulation. Therefore, although the myoepithelial sheath has smooth muscle-like contractile apparatuses, it has a striated muscle-like regulatory mechanism through troponin/tropomyosin. Here we report that the myoepithelial sheath has a distinct myosin population containing nonmuscle myosin II isoforms, which is regulated by phosphorylation and essential for ovulation. MLC-4, a nonmuscle myosin regulatory light chain, localizes to small punctate structures and does not colocalize with large, needle-like myosin filaments containing MYO-3, a striated-muscle myosin isoform. RNA interference of MLC-4, as well as of its upstream regulators, LET-502 (Rho-associated coiled-coil forming kinase) and MEL-11 (a myosin-binding subunit of myosin phosphatase), impairs ovulation. Expression of a phosphomimetic MLC-4 mutant mimicking a constitutively active state also impairs ovulation. A striated-muscle myosin (UNC-54) appears to provide partially compensatory contractility. Thus the results indicate that the two spatially distinct myosin II populations coordinately regulate ovulatory contraction of the myoepithelial sheath. PMID:26864628

  3. Effects of pathogenic proline mutations on myosin assembly.

    PubMed

    Buvoli, Massimo; Buvoli, Ada; Leinwand, Leslie A

    2012-02-01

    Laing distal myopathy (MPD1) is a genetically dominant myopathy characterized by early and selective weakness of the distal muscles. Mutations in the MYH7 gene encoding for the β-myosin heavy chain are the underlying genetic cause of MPD1. However, their pathogenic mechanisms are currently unknown. Here, we measure the biological effects of the R1500P and L1706P MPD1 mutations in different cellular systems. We show that, while the two mutations inhibit myosin self-assembly in non-muscle cells, they do not prevent incorporation of the mutant myosin into sarcomeres. Nevertheless, we find that the L1706P mutation affects proper antiparallel myosin association by accumulating in the bare zone of the sarcomere. Furthermore, bimolecular fluorescence complementation assay shows that the α-helix containing the R1500P mutation folds into homodimeric (mutant/mutant) and heterodimeric [mutant/wild type (WT)] myosin molecules that are competent for sarcomere incorporation. Both mutations also form aggregates consisting of cytoplasmic vacuoles surrounding paracrystalline arrays and amorphous rod-like inclusions that sequester WT myosin. Myosin aggregates were also detected in transgenic nematodes expressing the R1500P mutation. By showing that the two MPD1 mutations can have dominant effects on distinct components of the contractile apparatus, our data provide the first insights into the pathogenesis of the disease. PMID:22155079

  4. The Dictyostelium class I myosin, MyoD, contains a novel light chain that lacks high-affinity calcium-binding sites.

    PubMed Central

    De La Roche, Marc A; Lee, Sheu-Fen; Côté, Graham P

    2003-01-01

    Dictyostelium discoideum MyoD, a long-tailed class I myosin, co-purified with two copies of a 16 kDa light chain. Sequence analysis of the MyoD light chain showed it to be a unique protein, termed MlcD, that shares 44% sequence identity with Dictyostelium calmodulin and 43% sequence identity with Acanthamoeba castellanii myosin IC light chain. MlcD comprises four EF-hands; however, EF-hands 2-4 contain mutations in key Ca2+-co-ordinating residues that would be predicted to impair Ca2+ binding. Electrospray ionization MS of MlcD in the presence of Ca2+ and La3+ showed the presence of one major and one minor metal-binding site. MlcD contains a single tryptophan residue (Trp39), the fluorescence intensity of which was quenched upon addition of Ca2+ or Mg2+, yielding apparent dissociation constants ( K'(d)) of 52 microM for Ca2+ and 450 microM for Mg2+. The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+. Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs. FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC. Through its specific association with the Ca2+-insensitive MlcD, MyoD may exhibit distinct regulatory properties that distinguish it from myosin I isoenzymes with calmodulin light chains. PMID:12826013

  5. Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction.

    PubMed

    Gregorich, Zachery R; Peng, Ying; Cai, Wenxuan; Jin, Yutong; Wei, Liming; Chen, Albert J; McKiernan, Susan H; Aiken, Judd M; Moss, Richard L; Diffee, Gary M; Ge, Ying

    2016-08-01

    Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly and thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously unreported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction and suggest that therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia. PMID:27362462

  6. The human myosin light chain kinase (MLCK) from hippocampus: Cloning, sequencing, expression, and localization to 3qcen-q21

    SciTech Connect

    Potier, M.C.; Rossier, J.; Turnell, W.G.; Pekarsky, Y.; Gardiner, K.

    1995-10-10

    Myosin light chain kinase (MLCK), a key enzyme in muscle contraction, has been shown by immunohistology to be present in neurons and glia. We describe here the cloning of the cDNA for human MLCK from hippocampus, encoding a protein sequence 95% similar to smooth muscle MLCKs but less than 60% similar to skeletal muscle MLCKs. The cDNA clone detected two RNA transcripts in human frontal and entorhinal cortex, in hippocampus, and in jejunum, one corresponding to MLCK and the other probably to telokin, the carboxy-terminal 154 codons of MLCK expressed as an independent protein in smooth muscle. Levels of expression were lower in brain compared to smooth muscle. We show that within the protein sequence, a motif of 28 or 24 residues is repeated five times, the second repeat ending with the putative methionine start codon. These repeats overlap with a second previously reported module of 12 residues repeated five times in the human sequence. In addition, the acidic C-terminus of all MLCKs from both brain and smooth muscle resembles the C-terminus of tubulins. The chromosomal localization of the gene for human MLCK is shown to be at 3qcen-q21, as determined by PCR and Southern blotting using two somatic cell hybrid panels. 33 refs., 8 figs.

  7. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase

    PubMed Central

    Bhargava, Amol; Cotton, James A.; Dixon, Brent R.; Gedamu, Lashitew; Yates, Robin M.; Buret, Andre G.

    2015-01-01

    Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections. PMID:26334299

  8. Gene expression patterns in transgenic mouse models of hypertrophic cardiomyopathy caused by mutations in myosin regulatory light chain.

    PubMed

    Huang, Wenrui; Kazmierczak, Katarzyna; Zhou, Zhiqun; Aguiar-Pulido, Vanessa; Narasimhan, Giri; Szczesna-Cordary, Danuta

    2016-07-01

    Using microarray and bioinformatics, we examined the gene expression profiles in transgenic mouse hearts expressing mutations in the myosin regulatory light chain shown to cause hypertrophic cardiomyopathy (HCM). We focused on two malignant RLC-mutations, Arginine 58→Glutamine (R58Q) and Aspartic Acid 166 → Valine (D166V), and one benign, Lysine 104 → Glutamic Acid (K104E)-mutation. Datasets of differentially expressed genes for each of three mutants were compared to those observed in wild-type (WT) hearts. The changes in the mutant vs. WT samples were shown as fold-change (FC), with stringency FC ≥ 2. Based on the gene profiles, we have identified the major signaling pathways that underlie the R58Q-, D166V- and K104E-HCM phenotypes. The correlations between different genotypes were also studied using network-based algorithms. Genes with strong correlations were clustered into one group and the central gene networks were identified for each HCM mutant. The overall gene expression patterns in all mutants were distinct from the WT profiles. Both malignant mutations shared certain classes of genes that were up or downregulated, but most similarities were noted between D166V and K104E mice, with R58Q hearts showing a distinct gene expression pattern. Our data suggest that all three HCM mice lead to cardiomyopathy in a mutation-specific manner and thus develop HCM through diverse mechanisms. PMID:26906074

  9. Myosin light chain phosphatase activation is involved in the hydrogen sulfide-induced relaxation in mouse gastric fundus.

    PubMed

    Dhaese, Ingeborg; Lefebvre, Romain A

    2009-03-15

    The relaxant effect of hydrogen sulfide (H(2)S) in the vascular tree is well established but its influence and mechanism of action in gastrointestinal smooth muscle was hardly investigated. The influence of H(2)S on contractility in mouse gastric fundus was therefore examined. Sodium hydrogen sulfide (NaHS; H(2)S donor) was administered to prostaglandin F(2alpha) (PGF(2alpha))-contracted circular muscle strips of mouse gastric fundus, before and after incubation with interfering drugs. NaHS caused a concentration-dependent relaxation of the pre-contracted mouse gastric fundus strips. The K(+) channels blockers glibenclamide, apamin, charybdotoxin, 4-aminopyridin and barium chloride had no influence on the NaHS-induced relaxation. The relaxation by NaHS was also not influenced by L-NAME, ODQ and SQ 22536, inhibitors of the cGMP and cAMP pathway, by nerve blockers capsazepine, omega-conotoxin and tetrodotoxin or by several channel and receptor blockers (ouabain, nifedipine, 2-aminoethyl diphenylborinate, ryanodine and thapsigargin). The myosin light chain phosphatase (MLCP) inhibitor calyculin-A reduced the NaHS-induced relaxation, but the Rho-kinase inhibitor Y-27632 had no influence. We show that NaHS is able to relax PGF(2alpha)-contracted mouse gastric fundus strips. The results suggest that in the mouse gastric fundus, H(2)S causes relaxation at least partially via activation of MLCP. PMID:19374871

  10. A mutation in the atrial-specific myosin light chain gene (MYL4) causes familial atrial fibrillation.

    PubMed

    Orr, Nathan; Arnaout, Rima; Gula, Lorne J; Spears, Danna A; Leong-Sit, Peter; Li, Qiuju; Tarhuni, Wadea; Reischauer, Sven; Chauhan, Vijay S; Borkovich, Matthew; Uppal, Shaheen; Adler, Arnon; Coughlin, Shaun R; Stainier, Didier Y R; Gollob, Michael H

    2016-01-01

    Atrial fibrillation (AF), the most common arrhythmia, is a growing epidemic with substantial morbidity and economic burden. Mechanisms underlying vulnerability to AF remain poorly understood, which contributes to the current lack of highly effective therapies. Recognizing mechanistic subtypes of AF may guide an individualized approach to patient management. Here, we describe a family with a previously unreported syndrome characterized by early-onset AF (age <35 years), conduction disease and signs of a primary atrial myopathy. Phenotypic penetrance was complete in all mutation carriers, although complete disease expressivity appears to be age-dependent. We show that this syndrome is caused by a novel, heterozygous p.Glu11Lys mutation in the atrial-specific myosin light chain gene MYL4. In zebrafish, mutant MYL4 leads to disruption of sarcomeric structure, atrial enlargement and electrical abnormalities associated with human AF. These findings describe the cause of a rare subtype of AF due to a primary, atrial-specific sarcomeric defect. PMID:27066836