scholarly journals Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. Implications for twitch potentiation in intact muscle.

1989 ◽  
Vol 93 (5) ◽  
pp. 855-883 ◽  
Author(s):  
J M Metzger ◽  
M L Greaser ◽  
R L Moss
Keyword(s):  
Steady State ◽  
Light Chain ◽  
Time Course ◽  
Myosin Light Chain ◽  
Isometric Force ◽  
Vastus Lateralis ◽  
Isometric Tension ◽  
National Academy Of Sciences ◽  
Cross Bridge ◽  
Myosin Light Chain 2

The Ca2+ sensitivities of the rate constant of tension redevelopment (ktr; Brenner, B., and E. Eisenberg. 1986. Proceedings of the National Academy of Sciences. 83:3542-3546) and isometric force during steady-state activation were examined as functions of myosin light chain 2 (LC2) phosphorylation in skinned single fibers from rabbit and rat fast-twitch skeletal muscles. To measure ktr the fiber was activated with Ca2+ and steady isometric tension was allowed to develop; subsequently, the fiber was rapidly (less than 1 ms) released to a shorter length and then reextended by approximately 200 nm per half sarcomere. This maneuver resulted in the complete dissociation of cross-bridges from actin, so that the subsequent redevelopment of tension was related to the rate of cross-bridge reattachment. The time course of tension redevelopment, which was recorded under sarcomere length control, was best fit by a first-order exponential equation (i.e., tension = C(1 - e-kt) to obtain the value of ktr. In control fibers, ktr increased sigmoidally with increases in [Ca2+]; maximum values of ktr were obtained at pCa 4.5 and were significantly greater in rat superficial vastus lateralis fibers (26.1 +/- 1.2 s-1 at 15 degrees C) than in rabbit psoas fibers (18.7 +/- 1.0 s-1). Phosphorylation of LC2 was accomplished by repeated Ca2+ activations (pCa 4.5) of the fibers in solutions containing 6 microM calmodulin and 0.5 microM myosin light chain kinase, a protocol that resulted in an increase in LC2 phosphorylation from approximately 10% in the control fibers to greater than 80% after treatment. After phosphorylation, ktr was unchanged at maximum or very low levels of Ca2+ activation. However, at intermediate levels of Ca2+ activation, between pCa 5.5 and 6.2, there was a significant increase in ktr such that this portion of the ktr-pCa relationship was shifted to the left. The steady-state isometric tension-pCa relationship, which in control fibers was left shifted with respect to the ktr-pCa relationship, was further left-shifted after LC2 phosphorylation. Phosphorylation of LC2 had no effect upon steady-state tension during maximum Ca2+ activation. In fibers from which troponin C was partially extracted to disrupt molecular cooperativity within the thin filament (Moss et al. 1985. Journal of General Physiology. 86:585-600), the effect of LC2 phosphorylation to increase the Ca2+ sensitivity of steady-state isometric force was no longer evident, although the effect of phosphorylation to increase ktr was unaffected by this maneuver.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Mavacamten Decreases Maximal Force and Ca2+-sensitivity in the N47K-Myosin Regulatory Light Chain Mouse Model of Hypertrophic Cardiomyopathy

2020 ◽  
Author(s):  
Peter O Awinda ◽  
Marissa Watanabe ◽  
Yemeserach M. Bishaw ◽  
Anna M Huckabee ◽  
Keinan B Agonias ◽  
...  
Keyword(s):  
Heart Disease ◽  
Hypertrophic Cardiomyopathy ◽  
Light Chain ◽  
Isometric Force ◽  
Regulatory Light Chain ◽  
Isometric Tension ◽  
Myosin Regulatory Light Chain ◽  
Cardiac Myosin ◽  
Cross Bridge ◽  
Obstructive Hypertrophic Cardiomyopathy

Morbidity and mortality associated with heart disease is a growing threat to the global population and novel therapies are needed. Mavacamten (formerly called MYK-461) is a small molecule that binds to cardiac myosin and inhibits myosin ATPase. Mavacamten is currently in clinical trials for the treatment of obstructive hypertrophic cardiomyopathy (HCM), and it may provide benefits for treating other forms of heart disease. We investigated the effect of mavacamten on cardiac muscle contraction in two transgenic mouse lines expressing the human isoform of cardiac myosin regulatory light chain (RLC) in their hearts. Control mice expressed wild-type RLC (WT-RLC), and HCM mice expressed the N47K RLC mutation. In the absence of mavacamten, skinned papillary muscle strips from WT-RLC mice produced greater isometric force than strips from N47K mice. Adding 0.3 µM mavacamten decreased maximal isometric force and reduced Ca2+-sensitivity of contraction for both genotypes, but this reduction in pCa50 was nearly twice as large for WT-RLC vs. N47K. We also used stochastic length-perturbation analysis to characterize cross-bridge kinetics. The cross-bridge detachment rate was measured as a function of [MgATP] to determine the effect of mavacamten on myosin nucleotide handling rates. Mavacamten increased the MgADP release and MgATP binding rates for both genotypes, thereby contributing to faster cross-bridge detachment, which could speed myocardial relaxation during diastole. Our data suggest that mavacamten reduces isometric tension and Ca2+-sensitivity of contraction via decreased strong cross-bridge binding. Mavacamten may become a useful therapy for patients with heart disease, including some forms of HCM.

Isoproterenol attenuates myosin phosphorylation and contraction of tracheal muscle

1989 ◽  
Vol 66 (5) ◽  
pp. 2017-2022 ◽  
Author(s):  
K. Obara ◽  
P. de Lanerolle
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Isometric Force ◽  
Smooth Muscles ◽  
Inhibitory Effect ◽  
Isometric Tension ◽  
Myosin Light Chain Phosphorylation ◽  
Response Curves ◽  
Shortening Velocity ◽  
Myosin Phosphorylation

The effects of isoproterenol on isometric force, unloaded shortening velocity, and myosin phosphorylation were examined in thin muscle bundles (0.1–0.2 mm diam) dissected from lamb tracheal smooth muscle. Methacholine (10(-6) M) induced rapid increases in isometric force and in phosphorylation of the 20,000-Da myosin light chain. Myosin phosphorylation remained elevated during steady-state maintenance of isometric force. The shortening velocity peaked at 15 s after stimulation with methacholine and then declined to approximately 45% of the maximal value by 3 min. Isoproterenol pretreatment inhibited methacholine-stimulated myosin light chain phosphorylation, shortening velocity, and force during the early stages of force generation. However, the inhibitory effect of isoproterenol on force and myosin phosphorylation is proportionally greater than that on shortening velocity. Isoproterenol pretreatment also caused a rightward non-parallel shift in the methacholine dose-response curves for both isometric tension and myosin light chain phosphorylation. These data demonstrate that isoproterenol attenuates the contractile properties of airway smooth muscles by affecting the rate and extent of myosin light chain phosphorylation, perhaps through a mechanism that involves the synergistic interaction of myosin light chain kinase phosphorylation and Ca2+ metabolism.

Substance P contracts bovine tracheal smooth muscle via activation of myosin light chain kinase

1990 ◽  
Vol 259 (2) ◽  
pp. C258-C265 ◽  
Author(s):  
M. A. Corson ◽  
J. R. Sellers ◽  
R. S. Adelstein ◽  
M. Schoenberg
Keyword(s):  
Substance P ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Time Course ◽  
Myosin Light Chain ◽  
Immunoblot Analysis ◽  
Isometric Tension ◽  
Peak Tension ◽  
Tension Response

At near-threshold substance P concentrations, the isometric tension response of bovine tracheal strips is almost completely abolished by atropine, indicating mediation of contraction via substance P-stimulated release of acetylcholine from prejunctional nerve terminals. At near-maximal concentrations, the atropine-inhibited component of the tension response is less than 25%, indicating mainly direct activation. Under conditions in which activation by substance P is direct, peak tension is reached in approximately 11 min. Immunoblot analysis of the time course of phosphorylation of the 20-kDa myosin light chain (LC20) reveals incorporation of approximately 0.5 mol phosphate/mol light chain at 10 min. Two-dimensional tryptic phosphopeptide analysis of phosphorylated light chain reveals a single major phosphopeptide. The peptide migrates identically with that produced by myosin light chain kinase phosphorylation of purified tracheal myosin in vitro. Contraction stimulated by acetylcholine is more rapid, with attainment of peak tension in 2.5 min and a peak LC20 phosphorylation of 0.65 mol/mol. These results indicate that 1) substance P mediates contraction of bovine trachea both directly and indirectly, and 2) under conditions in which activation is direct, the tension and phosphorylation responses qualitatively resemble those observed with acetylcholine.

Rapid turnover of myosin light chain phosphate during cross-bridge cycling in smooth muscle

1994 ◽  
Vol 267 (4) ◽  
pp. C1160-C1166 ◽  
Author(s):  
T. M. Butler ◽  
S. R. Narayan ◽  
S. U. Mooers ◽  
M. J. Siegman
Keyword(s):  
Smooth Muscle ◽  
Rate Constant ◽  
Light Chain ◽  
Time Course ◽  
Myosin Light Chain ◽  
Specific Activity ◽  
Energy Requirement ◽  
High Specific Activity ◽  
Cross Bridge ◽  
Muscle Myosin

The rate of phosphatase-mediated dephosphorylation of the regulatory light chain of smooth muscle myosin was determined under nearly steady-state conditions in permeabilized muscles, from the time course of incorporation of 33P-labeled phosphate into the light chain after the photolytic release of [gamma-33P]ATP from high specific activity caged [gamma-33P]ATP. The extent of myosin light chain phosphorylation is unchanged, and, if the kinase and phosphatase reactions are irreversible, the rate constant for the exponential increase in 33P in the light chain is equal to the rate constant for the phosphatase reaction. Under activated conditions (pCa 4.5) at 20 degrees C, the incorporation of 33P into approximately 80% of the phosphorylated light chain is fit by a single exponential with a rate constant of 0.37 s-1. ATP usage due to phosphorylation and dephosphorylation of the light chain is about one-third of the suprabasal energy requirement. The high phosphatase rate constant suggests that dephosphorylation of the light chain is rapid enough to interact with and potentially modify the completion of the cross-bridge cycle.

Myosin light chain phosphorylation associated with contraction in arterial smooth muscle

1981 ◽  
Vol 240 (5) ◽  
pp. C222-C233 ◽  
Author(s):  
S. P. Driska ◽  
M. O. Aksoy ◽  
R. A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Isometric Force ◽  
Physiological Salt Solution ◽  
Physiological Control ◽  
The Media ◽  
Low Levels ◽  
State Force

The hypothesis that Ca2+ initiates contraction in smooth muscle by activating an endogenous myosin light chain kinase (MLCK) that phosphorylates the 20,000 dalton light chain (LC 20) of myosin was tested in tissues prepared from the media of swine carotid arteries. Unstimulated tissues with low levels of tone exhibited low levels of phosphorylated LC 20. On stimulation with a high-K+ physiological salt solution containing 1.6 mM CaCl2, LC 20 phosphorylation increased to 0.6 mol P/mol LC 20 within 30 s. This increase preceded force development, which required 2-4 min to attain a maximum steady-state value of 3.34 +/- 0.15 (SE) X 10(5) N/m2. These results support the hypothesis, as the stimulus was submaximal for the preparation. However, LC 20 phosphorylation declined significantly from its peak value before steady-state force was attained, reaching near control levels after 10 min of stimulation. The results suggest that Ca2+-stimulated LC 20 phosphorylation is an important physiological control mechanism but that additional factors are involved in the maintenance of tonic isometric force.

Smooth muscle energetics and theories of cross-bridge regulation

1990 ◽  
Vol 258 (2) ◽  
pp. C369-C375 ◽  
Author(s):  
R. J. Paul
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Steady State ◽  
Time Course ◽  
Isometric Force ◽  
Muscle Energetics ◽  
Cross Bridge ◽  
Tension Cost ◽  
Low Tension ◽  
Smooth Muscle Energetics

The energetics of smooth muscle is characterized by low tension cost (rate of ATP utilization per isometric force/cross-section area), ranging from 100- to 500-fold less than skeletal muscle. The efficiency (ATP usage per work) of smooth muscle, although less well documented, is also somewhat (4-fold) less than skeletal muscle. Another well-known characteristic of smooth muscle is the linear relation between the steady-state of ATP utilization (JATP) and isometric force. Recently, Murphy and colleagues [C.-M. Hai and R. A. Murphy. Am. J. Physiol. 254 (Cell Physiol. 23) C99-C106, 1988] have put forth a kinetic model of cross-bridge regulation that predicts the time course of stress and myosin light chain phosphorylation (MLC-Pi). The energetics consequences of this model, in brief, are that the low tension cost is partly attributed to a slow detachment rate of the myosin cross bridge when dephosphorylated when attached to actin ("latch state"), whereas the lower efficiency is ascribed to a high rate of myosin phosphorylation-dephosphorylation inherent to a fit of data to this kinetic scheme. This latter corollary is somewhat controversial in light of current interpretations of smooth muscle energetics data. Using SCoP software (National Biomedical Simulation Resource, Duke University), we tested this model in terms of fitting existing data with respect to 1) is a high myosin-dephosphorylation adenosine triphosphatase (ATPase) necessary to fit the available data on the time course of stress and MLC-Pi?; and 2) can this model predict the observed linear relation between the steady-state rate of ATP hydrolysis (JATP) and isometric force?(ABSTRACT TRUNCATED AT 250 WORDS)

Halothane inhibits agonist-induced potentiation of rMLC phosphorylation in permeabilized airway smooth muscle

1997 ◽  
Vol 273 (1) ◽  
pp. L80-L85 ◽  
Author(s):  
K. A. Jones ◽  
A. Hirasaki ◽  
D. H. Bremerich ◽  
C. Jankowski ◽  
D. O. Warner
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Vascular Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Isometric Force ◽  
State Level ◽  
Steady State Level ◽  
Regulatory Myosin Light Chain ◽  
The Relationship

Agonist-induced increases in CA2+ sensitivity are mediated in part by mechanisms that increase phosphorylation of the regulatory myosin light chain (rMLC) at constant cytosolic Ca2+ concentration ([Ca2+]i). The current study tested the hypothesis that halothane inhibits acetylcholine (ACh)-induced potentiation of rMLC phosphorylation in beta-escin-permeabilized canine tracheal smooth muscle. ACh plus GTP significantly potentiated the increase in isometric force and rMLC phosphorylation induced by 0.8 microM free Ca2+. However, whereas the potentiation of isometric force was sustained, the potentiation of rMLC phosphorylation was biphasic, peaking at 0.5 min and then declining by approximately 10 min to a steady-state level significantly above that induced by 0.8 microM free Ca2+ alone. This finding suggests that mechanisms in addition to changes in rMLC phosphorylation may mediate ACh-induced Ca2+ sensitization, as has been reported for vascular smooth muscle. Halothane (0.91 +/- 0.10 mM) significantly inhibited ACh plus GTP-induced potentiation of rMLC phosphorylation and isometric force after 2 (peak rMLC phosphorylation) and 15 (steady-state rMLC phosphorylation) min of stimulation. However, the effect of halothane on the potentiation of isometric force was significantly less than that expected from its effect on rMLC phosphorylation (i.e., halothane changed the relationship between rMLC phosphorylation and isometric force). These results demonstrate that halothane inhibits the ACh-induced increase in Ca2+ sensitivity by inhibiting the membrane receptor-coupled mechanisms that increase rMLC phosphorylation at constant submaximal [Ca2+]i. Possible additional effects of halothane on rMLC phosphorylation-independent mechanisms cannot be ruled out.

Role of Ca2+ and myosin light chain phosphorylation in regulation of smooth muscle

1982 ◽  
Vol 242 (1) ◽  
pp. C109-C116 ◽  
Author(s):  
M. O. Aksoy ◽  
R. A. Murphy ◽  
K. E. Kamm
Keyword(s):  
Light Chain ◽  
Time Course ◽  
Myosin Light Chain ◽  
Carotid Arteries ◽  
Stable Steady State ◽  
Shortening Velocity ◽  
Relaxation Cycle ◽  
Cross Bridge ◽  
Contraction And Relaxation

The time course of phosphorylation of the 20,000-dalton myosin light chain (LC 20) was determined during contraction and relaxation in K+- and histamine-stimulated medial strips of swine carotid arteries. Resting LC 20 phosphorylation levels of 0.15 mol P/mol LC 20 rapidly increased to peak values of 0.6-0.7 mol P/mol LC 20 after stimulation and then declined significantly, although stress continued to rise to a stable steady-state maximum. LC 20 dephosphorylation after agonist washout preceded the decline in isometric stress. Over the entire contraction-relaxation cycle, phosphorylation was correlated with shortening velocity and not with developed stress. The maximum shortening velocity with no external load (Vo) was directly proportional to LC 20 phosphorylation (r = 0.986). The data indicate that LC 20 phosphorylation is necessary for cross-bridge cycling leading to shortening or stress development but that stress can be maintained by additional mechanisms. We suggest that dephosphorylation of an attached cross bridge in the presence of Ca2+ arrests the cycle, forming an attached, noncycling cross bridge.

Latch-bridge model in smooth muscle: [Ca2+]i can quantitatively predict stress

1990 ◽  
Vol 259 (2) ◽  
pp. C251-C257 ◽  
Author(s):  
C. M. Rembold ◽  
R. A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Time Course ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Force Production ◽  
Cross Bridge ◽  
Bridge Model ◽  
Latch State

Ca2+ concentration ([Ca2+])-dependent cross-bridge phosphorylation by myosin light chain kinase is postulated to be the primary regulator of stress development in smooth muscle. A four-state model of cross-bridge function, regulated only by [Ca2+]-dependent changes in myosin kinase activity, has been proposed to explain contraction and the latch state of smooth muscle (high force with reduced cross-bridge cycling and ATP consumption). A key test of this model is to determine whether changes in myoplasmic [Ca2+], per se, can quantitatively predict changes in myosin kinase activity, cross-bridge phosphorylation, and therefore force production. We find that changes in aequorin-estimated myoplasmic [Ca2+] can quantitatively predict the time course of phosphorylation and isometric stress production in response to stimulation with histamine and angiotensin II and during adenosine 3',5'-cyclic monophosphate-mediated relaxation when [Ca2+] is not changing rapidly. These results suggest that changes in myoplasmic [Ca2+] and activation of myosin light chain kinase may be sufficient to explain both contraction and relaxation of agonist stimulated swine carotid arterial smooth muscle.

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