scholarly journals Myosin light chain phosphorylation facilitates in vivo myosin filament reassembly after mechanical perturbation

2002 ◽  
Vol 282 (6) ◽  
pp. C1298-C1305 ◽  
Author(s):  
D. Qi ◽  
R. W. Mitchell ◽  
T. Burdyga ◽  
L. E. Ford ◽  
K.-H. Kuo ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Isometric Force ◽  
Thick Filament ◽  
Myosin Filament ◽  
Mechanical Perturbation ◽  
Cross Sectional ◽  
Thick Filaments ◽  
Mlc Phosphorylation

Phosphorylation of the 20-kDa regulatory myosin light chain (MLC) of smooth muscle is known to cause monomeric myosins in solution to self-assemble into thick filaments. The role of MLC phosphorylation in thick filament formation in intact muscle, however, is not clear. It is not known whether the phosphorylation is necessary to initiate thick filament assembly in vivo. Here we show, by using a potent inhibitor of MLC kinase (wortmannin), that the MLC phosphorylation and isometric force in trachealis muscle could be abolished without affecting calcium transients. By measuring cross-sectional densities of the thick filaments electron microscopically, we also show that inhibition of MLC phosphorylation alone did not cause disassembly of the filaments. The unphosphorylated thick filaments, however, partially dissolved when the muscle was subjected to oscillatory strains (which caused a 25% decrease in the thick filament density). The postoscillation filament density recovered to the preoscillation level only when wortmannin was removed and the muscle was stimulated. The data suggest that in vivo thick filament reassembly after mechanical perturbation is facilitated by the cyclic MLC phosphorylation associated with repeated stimulation.

Morphometry and muscle gene expression in hypertrophied hearts from polyomavirus large T antigen transgenic mice

1995 ◽  
Vol 269 (1) ◽  
pp. H86-H95 ◽  
Author(s):  
E. Holder ◽  
B. Mitmaker ◽  
L. Alpert ◽  
L. Chalifour
Keyword(s):  
Transgenic Mice ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Troponin I ◽  
T Antigen ◽  
Large T Antigen ◽  
Cross Sectional

Transgenic mice expressing polyomavirus large T antigen (PVLT) in cardiomyocytes develop a cardiac hypertrophy in adulthood. Morphometric analysis identified cardiomyocytes enlarged up to ninefold in cross-sectional area in the adult transgenic hearts compared with normal age-matched nontransgenic hearts. Most enlarged cardiomyocytes were found in the subendocardium, whereas normal-sized cardiomyocytes were localized to the midmyocardium. Transgenic hearts did not express detectable skeletal muscle actin mRNA or protein, or skeletal troponin I isoform mRNA. Some, but not all, transgenic hearts expressed an increase in the beta-myosin heavy chain mRNA. All five transgenic mice tested had increased expression of atrial natriuretic factor (ANF) mRNA. Whereas normal hearts expressed three myosin light chain proteins of 19, 16, and 15 kDa, we found that the 19-kDa myosin light chain was not observed in the transgenic hearts. We conclude that adult, PVLT-expressing, transgenic mice developed enlarged cardiomyocytes with an increase in beta-myosin heavy chain and ANF mRNA expression, but a widespread skeletal isoform usage was not present in these transgenic mice. The adult transgenic hearts thus display histological and molecular changes similar to those found in hypertrophy induced by a pressure overload in vivo.

Influence of calcium on myosin thick filament formation in intact airway smooth muscle

2002 ◽  
Vol 282 (2) ◽  
pp. C310-C316 ◽  
Author(s):  
Ana M. Herrera ◽  
Kuo-Hsing Kuo ◽  
Chun Y. Seow
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Calcium Level ◽  
Muscle Activation ◽  
Isometric Force ◽  
Smooth Muscles ◽  
Thick Filament ◽  
Filament Formation ◽  
Cross Sectional ◽  
Thick Filaments

Myosin thick filaments have been shown to be structurally labile in intact smooth muscles. Although the mechanism of thick filament assembly/disassembly for purified myosins in solution has been well described, regulation of thick filament formation in intact muscle is still poorly understood. The present study investigates the effect of resting calcium level on thick filament maintenance in intact airway smooth muscle and on thick filament formation during activation. Cross-sectional density of the thick filaments measured electron microscopically showed that the density increased substantially (144%) when the muscle was activated. The abundance of filamentous myosins in relaxed muscle was calcium sensitive; in the absence of calcium (with EGTA), the filament density deceased by 35%. Length oscillation imposed on the muscle under zero-calcium conditions produced no further reduction in the density. Isometric force and filament density recovered fully after reincubation of the muscle in normal physiological saline. The results suggest that in airway smooth muscle, filamentous myosins exist in equilibrium with monomeric myosins; muscle activation favors filament formation, and the resting calcium level is crucial for preservation of the filaments in the relaxed state.

scholarly journals Effects of phosphorylation by myosin light chain kinase on the structure of Limulus thick filaments.

1991 ◽  
Vol 113 (3) ◽  
pp. 563-572 ◽  
Author(s):  
R J Levine ◽  
P D Chantler ◽  
R W Kensler ◽  
J L Woodhead
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Preceding Paper ◽  
Thick Filament ◽  
Light Chains ◽  
Helical Surface ◽  
Regulatory Light Chains ◽  
Thick Filaments ◽  
Cell Biol

The results discussed in the preceding paper (Levine, R. J. C., J. L. Woodhead, and H. A. King. 1991. J. Cell Biol. 113:563-572.) indicate that A-band shortening in Limulus muscle is a thick filament response to activation that occurs largely by fragmentation of filament ends. To assess the effect of biochemical changes directly associated with activation on the length and structure of thick filaments from Limulus telson muscle, a dually regulated tissue (Lehman, W., J. Kendrick-Jones, and A. G. Szent Gyorgyi. 1973. Cold Spring Harbor Symp. Quant. Biol. 37:319-330.) we have examined the thick filament response to phosphorylation of myosin regulatory light chains. In agreement with the previous work of J. Sellers (1981. J. Biol. Chem. 256:9274-9278), Limulus myosin, incubated with partially purified chicken gizzard myosin light chain kinase (MLCK) and [gamma 32P]-ATP, binds 2 mol phosphate/mole protein. On autoradiographs of SDS-PAGE, the label is restricted to the two regulatory light chains, LC1 and LC2. Incubation of long (greater than or equal to 4.0 microns) thick filaments, separated from Limulus telson muscle under relaxing conditions, with either intact MLCK in the presence of Ca2+ and calmodulin, or Ca2(+)-independent MLCK obtained by brief chymotryptic digestion (Walsh, M. P., R. Dabrowska, S. Hinkins, and D. J. Hartshorne. 1982. Biochemistry. 21:1919-1925), causes significant changes in their structure. These include: disordering of the helical surface arrangement of myosin heads as they move away from the filament backbone; the presence of distal bends and breaks, with loss of some surface myosin molecules, in each polar filament half; and the production of shorter filaments and end-fragments. The latter structures are similar to those produced by Ca2(+)-activation of skinned fibers (Levine, R. J. C., J. L. Woodhead, and H. A. King. J. Cell Biol. 113:563-572). Rinsing experimental filament preparations with relaxing solution before staining restores some degree of order of the helical surface array, but not filament length. We propose that outward movement of myosin heads and thick filament shortening in Limulus muscle are responses to activation that are dependent on phosphorylation of regulatory myosin light chains. Filament shortening may be due, in large part, to breakage at the filament ends.

scholarly journals Effect of 17 days of bed rest on peak isometric force and unloaded shortening velocity of human soleus fibers

1997 ◽  
Vol 273 (5) ◽  
pp. C1690-C1699 ◽  
Author(s):  
J. J. Widrick ◽  
J. G. Romatowski ◽  
J. L. W. Bain ◽  
S. W. Trappe ◽  
T. A. Trappe ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Isometric Force ◽  
Bed Rest ◽  
Type I ◽  
Adult Males ◽  
Shortening Velocity ◽  
Cross Sectional ◽  
Myosin Heavy Chain Isoform ◽  
Type I Fibers

The purpose of this study was to examine the effect of prolonged bed rest (BR) on the peak isometric force (Po) and unloaded shortening velocity ( V o) of single Ca2+-activated muscle fibers. Soleus muscle biopsies were obtained from eight adult males before and after 17 days of 6° head-down BR. Chemically permeabilized single fiber segments were mounted between a force transducer and position motor, activated with saturating levels of Ca2+, and subjected to slack length steps. V owas determined by plotting the time for force redevelopment vs. the slack step distance. Gel electrophoresis revealed that 96% of the pre- and 87% of the post-BR fibers studied expressed only the slow type I myosin heavy chain isoform. Fibers with diameter >100 μm made up only 14% of this post-BR type I population compared with 33% of the pre-BR type I population. Consequently, the post-BR type I fibers ( n = 147) were, on average, 5% smaller in diameter than the pre-BR type I fibers ( n = 218) and produced 13% less absolute Po. BR had no overall effect on Po per fiber cross-sectional area (Po/CSA), even though half of the subjects displayed a decline of 9–12% in Po/CSA after BR. Type I fiber V oincreased by an average of 34% with BR. Although the ratio of myosin light chain 3 to myosin light chain 2 also rose with BR, there was no correlation between this ratio and V o for either the pre- or post-BR fibers. In separate fibers obtained from the original biopsies, quantitative electron microscopy revealed a 20–24% decrease in thin filament density, with no change in thick filament density. These results raise the possibility that alterations in the geometric relationships between thin and thick filaments may be at least partially responsible for the elevated V o of the post-BR type I fibers.

scholarly journals Postnatal maturation modulates relationships among cytosolic Ca2+, myosin light chain phosphorylation, and contractile tone in ovine cerebral arteries

2007 ◽  
Vol 293 (4) ◽  
pp. H2183-H2192 ◽  
Author(s):  
Renan J. Sandoval ◽  
Elisha R. Injeti ◽  
William T. Gerthoffer ◽  
William J. Pearce
Keyword(s):  
G Protein ◽  
Light Chain ◽  
Thin Filament ◽  
Myosin Light Chain ◽  
Cerebral Arteries ◽  
Thick Filament ◽  
Receptor Activation ◽  
Postnatal Maturation ◽  
Age Related ◽  
Mlc Phosphorylation

The present study tests the hypothesis that age-related changes in patterns of agonist-induced myofilament Ca2+ sensitization involve corresponding differences in the relative contributions of thick- and thin-filament regulation to overall myofilament Ca2+ sensitivity. Posterior communicating cerebral arteries from term fetal and nonpregnant adult sheep were used in measurements of cytosolic Ca2+, myosin light chain (MLC) phosphorylation, and contractile tensions induced by varying concentrations of K+ or serotonin [5-hydroxytryptamine (5-HT)]. The results were used to assess the relative contributions of the relationships between cytosolic Ca2+ and MLC phosphorylation (thick-filament reactivity), along with the relationships between MLC phosphorylation and contractile tension (thin-filament reactivity), to overall myofilament Ca2+ sensitivity. For K+-induced contractions, both fetal and adult arteries exhibited similar basal myofilament Ca2+ sensitivity. Despite this similarity, thick-filament reactivity was greater in fetal arteries, whereas thin-filament reactivity was greater in adult arteries. In contrast, 5-HT-induced contractions exhibited increased myofilament Ca2+ sensitivity compared with K+-induced contractions for both fetal and adult cerebral arteries, and the magnitude of this effect was greater in fetal compared with adult arteries. When interpreted together with our previous studies of 5-HT-induced myofilament Ca2+ sensitization, we attributed the present effects to agonist enhancement of thick-filament reactivity in fetal arteries mediated by G protein receptor activation of a PKC-independent but RhoA-dependent pathway. In adult arteries, agonist stimulation enhanced thin-filament reactivity was also probably mediated through G protein-coupled activation of RhoA-dependent and PKC-independent mechanisms. Overall, the present data demonstrate that agonist-enhanced myofilament Ca2+ sensitivity can be partitioned into separate thick- and thin-filament effects, the magnitudes of which are different between fetal and adult cerebral arteries.

scholarly journals FFAR1 activation attenuates histamine-induced myosin light chain phosphorylation and cortical tension development in human airway smooth muscle cells

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Shengjie Xu ◽  
Anthony Schwab ◽  
Nikhil Karmacharya ◽  
Gaoyuan Cao ◽  
Joanna Woo ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Airway Hyperreactivity ◽  
Human Airway Smooth Muscle ◽  
Tension Development ◽  
Cortical Tension ◽  
Human Airway ◽  
Mlc Phosphorylation

Abstract Background Activation of free fatty acid receptors (FFAR1 and FFAR4) which are G protein-coupled receptors (GPCRs) with established (patho)physiological roles in a variety of obesity-related disorders, induce human airway smooth muscle (HASM) cell proliferation and shortening. We reported amplified agonist-induced cell shortening in HASM cells obtained from obese lung donors. We hypothesized that FFAR1 modulate excitation–contraction (EC) coupling in HASM cells and play a role in obesity-associated airway hyperresponsiveness. Methods In HASM cells pre-treated (30 min) with FFAR1 agonists TAK875 and GW9508, we measured histamine-induced Ca2+ mobilization, myosin light chain (MLC) phosphorylation, and cortical tension development with magnetic twisting cytometry (MTC). Phosphorylation of MLC phosphatase and Akt also were determined in the presence of the FFAR1 agonists or vehicle. In addition, the effects of TAK875 on MLC phosphorylation were measured in HASM cells desensitized to β2AR agonists by overnight salmeterol treatment. The inhibitory effect of TAK875 on MLC phosphorylation was compared between HASM cells from age and sex-matched non-obese and obese human lung donors. The mean measurements were compared using One-Way ANOVA with Dunnett’s test for multiple group comparisons or Student’s t-test two-group comparison. For cortical tension measurements by magnetic twisted cytometry, mixed effect model using SAS V.9.2 was applied. Means were considered significant when p ≤ 0.05. Results Unexpectedly, we found that TAK875, a synthetic FFAR1 agonist, attenuated histamine-induced MLC phosphorylation and cortical tension development in HASM cells. These physiological outcomes were unassociated with changes in histamine-evoked Ca2+ flux, protein kinase B (AKT) activation, or MLC phosphatase inhibition. Of note, TAK875-mediated inhibition of MLC phosphorylation was maintained in β2AR-desensitized HASM cells and across obese and non-obese donor-derived HASM cells. Conclusions Taken together, our findings identified the FFAR1 agonist TAK875 as a novel bronchoprotective agent that warrants further investigation to treat difficult-to-control asthma and/or airway hyperreactivity in obesity.

scholarly journals Blockade of ROCK inhibits migration of human primary keratinocytes and malignant epithelial skin cells by regulating actomyosin contractility

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Srisathya Srinivasan ◽  
Sreya Das ◽  
Vishakha Surve ◽  
Ankita Srivastava ◽  
Sushant Kumar ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Focal Adhesions ◽  
Primary Keratinocytes ◽  
Skin Cells ◽  
Physiological Processes ◽  
Actomyosin Contractility ◽  
Malignant Skin ◽  
And Migration ◽  
Mlc Phosphorylation

AbstractActomyosin contractility, crucial for several physiological processes including migration, is controlled by the phosphorylation of myosin light chain (MLC). Rho-associated protein kinase (ROCK) and Myosin light chain kinase (MLCK) are predominant kinases that phosphorylate MLC. However, the distinct roles of these kinases in regulating actomyosin contractility and their subsequent impact on the migration of healthy and malignant skin cells is poorly understood. We observed that blockade of ROCK in healthy primary keratinocytes (HPKs) and epidermal carcinoma cell line (A-431 cells) resulted in loss of migration, contractility, focal adhesions, stress fibres, and changes in morphology due to reduction in phosphorylated MLC levels. In contrast, blockade of MLCK reduced migration, contractile dynamics, focal adhesions and phosphorylated MLC levels of HPKs alone and had no effect on A-431 cells due to the negligible MLCK expression. Using genetically modified A-431 cells expressing phosphomimetic mutant of p-MLC, we show that ROCK dependent phosphorylated MLC controls the migration, focal adhesion, stress fibre organization and the morphology of the cells. In conclusion, our data indicate that ROCK is the major kinase of MLC phosphorylation in both HPKs and A-431 cells, and regulates the contractility and migration of healthy as well as malignant skin epithelial cells.

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

2015 ◽  
Vol 93 (1) ◽  
pp. 23-32 ◽  
Author(s):  
William Gittings ◽  
Harish Aggarwal ◽  
James T. Stull ◽  
Rene Vandenboom
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Isometric Force ◽  
Conditioning Stimulus ◽  
Mouse Muscle ◽  
Tetanic Force ◽  
Concentric Force ◽  
Skeletal Myosin ◽  
Contraction Type

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.

Abstract P780: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Perike Srikanth ◽  
Andrielle E Capote ◽  
Alsina Katherina M ◽  
Benjamin Levin ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S.of 6.1 million. AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light-chain phosphorylation in AF. Objective: We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light-chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A), PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and an increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

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