Smooth, cardiac and skeletal muscle myosin force and motion generation assessed by cross-bridge mechanical interactions in vitro

1994 ◽  
Vol 15 (1) ◽  
pp. 11-19 ◽  
Author(s):  
D. E. Harris ◽  
S. S. Work ◽  
R. K. Wright ◽  
N. R. Alpert ◽  
D. M. Warshaw
Keyword(s):  
Skeletal Muscle ◽  
Motion Generation ◽  
Skeletal Muscle Myosin ◽  
Cross Bridge ◽  
Force And Motion ◽  
Muscle Myosin

scholarly journals ATP-dependent movement of myosin in vitro: characterization of a quantitative assay.

1984 ◽  
Vol 99 (5) ◽  
pp. 1867-1871 ◽  
Author(s):  
M P Sheetz ◽  
R Chasan ◽  
J A Spudich
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Quantitative Assay ◽  
Vitro Assay ◽  
Skeletal Muscle Myosin ◽  
In Vitro Characterization ◽  
Actin Cables ◽  
Muscle Myosin

Sheetz and Spudich (1983, Nature (Lond.), 303:31-35) showed that ATP-dependent movement of myosin along actin filaments can be measured in vitro using myosin-coated beads and oriented actin cables from Nitella. To establish this in vitro movement as a quantitative assay and to understand better the basis for the movement, we have defined the factors that affect the myosin-bead velocity. Beads coated with skeletal muscle myosin move at a rate of 2-6 micron/s, depending on the myosin preparation. This velocity is independent of myosin concentration on the bead surface for concentrations above a critical value (approximately 20 micrograms myosin/2.5 X 10(9) beads of 1 micron in diameter). Movement is optimal between pH 6.8 and 7.5, at KCl concentrations less than 70 mM, at ATP concentrations greater than 0.1 mM, and at Mg2+ concentrations between 2 and 6 mM. From the temperature dependence of bead velocity, we calculate activation energies of 90 kJ/mol below 22 degrees C and 40 kJ/mol above 22 degrees C. Different myosin species move at their own characteristic velocities, and these velocities are proportional to their actin-activated ATPase activities. Further, the velocities of beads coated with smooth or skeletal muscle myosin correlate well with the known in vivo rates of myosin movement along actin filaments in these muscles. This in vitro assay, therefore, provides a rapid, reproducible method for quantitating the ATP-dependent movement of myosin molecules on actin.

In Vitro Motility of Skeletal Muscle Myosin and Its Proteolytic Fragments1

1990 ◽  
Vol 107 (5) ◽  
pp. 671-679 ◽  
Author(s):  
Kingo Takiguchi ◽  
Hiroshi Hayashi ◽  
Eiji Kurimoto ◽  
Sugie Higasshi-Fujime
Keyword(s):  
Skeletal Muscle ◽  
Skeletal Muscle Myosin ◽  
In Vitro Motility ◽  
Muscle Myosin

Troponin in embryonic chick skeletal muscle cell in vitro: an immunoelectron microscopic study

1978 ◽  
Vol 36 (2) ◽  
pp. 162-163
Author(s):  
Yutaka Shimada ◽  
Takashi Obinata
Keyword(s):  
Skeletal Muscle ◽  
Chicken Breast ◽  
Thin Filaments ◽  
Structural Localization ◽  
Adult Chicken ◽  
Skeletal Muscle Myosin ◽  
Ammonium Sulphate Fractionation ◽  
The Right ◽  
Muscle Myosin

In developing skeletal muscle, myosin and actin are synthesized and polymerized into filamentous forms, thick and thin filaments, respectively. These myofilaments of two varieties have been shown, with the use of "decoration with heavy meromyosin" technique, to be arranged at the right polarity and spatial position as those seen in mature myofibrils from the initial phases of myofibrillogenesis. The question arises as to whether regulatory proteins are distributed along embryonic thin filaments from such early stages of development. In order to clarify this problem, the fine structural localization of troponin was investigated by the use of immunoelectron microscopy.Troponin and its components (troponin C [TN-C], I [TN-I] and T [TN-T]) were prepared from adult chicken breast muscles. Rabbit antisera against each of these troponin components were prepared. The serum was fractioned by ethanol or ammonium sulphate fractionation. Myogenic cells from 12-day chick embryonic thigh muscles were grown in monolayer and, after 2-8 days in vitro, the cultures were treated as follows: (i) They were immersed in 50% glycerol. The suspension of separated thin filaments was obtained by gentle homogenizing the cells.

scholarly journals Effect of low pH on single skeletal muscle myosin mechanics and kinetics

2008 ◽  
Vol 295 (1) ◽  
pp. C173-C179 ◽  
Author(s):  
E. P. Debold ◽  
S. E. Beck ◽  
D. M. Warshaw
Keyword(s):  
Skeletal Muscle ◽  
Single Molecule ◽  
Low Ph ◽  
Muscular Fatigue ◽  
Step Size ◽  
Skeletal Muscle Myosin ◽  
In Vitro Motility ◽  
Adp Release ◽  
Muscle Myosin

Acidosis (low pH) is the oldest putative agent of muscular fatigue, but the molecular mechanism underlying its depressive effect on muscular performance remains unresolved. Therefore, the effect of low pH on the molecular mechanics and kinetics of chicken skeletal muscle myosin was studied using in vitro motility (IVM) and single molecule laser trap assays. Decreasing pH from 7.4 to 6.4 at saturating ATP slowed actin filament velocity ( Vactin) in the IVM by 36%. Single molecule experiments, at 1 μM ATP, decreased the average unitary step size of myosin ( d) from 10 ± 2 nm (pH 7.4) to 2 ± 1 nm (pH 6.4). Individual binding events at low pH were consistent with the presence of a population of both productive (average d = 10 nm) and nonproductive (average d = 0 nm) actomyosin interactions. Raising the ATP concentration from 1 μM to 1 mM at pH 6.4 restored d (9 ± 3 nm), suggesting that the lifetime of the nonproductive interactions is solely dependent on the [ATP]. Vactin, however, was not restored by raising the [ATP] (1–10 mM) in the IVM assay, suggesting that low pH also prolongs actin strong binding ( ton). Measurement of ton as a function of the [ATP] in the single molecule assay suggested that acidosis prolongs ton by slowing the rate of ADP release. Thus, in a detachment limited model of motility (i.e., Vactin ∼ d/ ton), a slowed rate of ADP release and the presence of nonproductive actomyosin interactions could account for the acidosis-induced decrease in Vactin, suggesting a molecular explanation for this component of muscular fatigue.

scholarly journals Cardiac Myosin Promotes Thrombin Generation and Coagulation In Vitro and In Vivo

2020 ◽  
Vol 40 (4) ◽  
pp. 901-913 ◽  
Author(s):  
Jevgenia Zilberman-Rudenko ◽  
Hiroshi Deguchi ◽  
Meenal Shukla ◽  
Yoshimasa Oyama ◽  
Jennifer N. Orje ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Tissue Type ◽  
Cardiac Myosin ◽  
Skeletal Muscle Myosin ◽  
Coated Surfaces ◽  
Muscle Myosin

Objective: Cardiac myosin (CM) is structurally similar to skeletal muscle myosin, which has procoagulant activity. Here, we evaluated CM’s ex vivo, in vivo, and in vitro activities related to hemostasis and thrombosis. Approach and Results: Perfusion of fresh human blood over CM-coated surfaces caused thrombus formation and fibrin deposition. Addition of CM to blood passing over collagen-coated surfaces enhanced fibrin formation. In a murine ischemia/reperfusion injury model, exogenous CM, when administered intravenously, augmented myocardial infarction and troponin I release. In hemophilia A mice, intravenously administered CM reduced tail-cut-initiated bleeding. These data provide proof of concept for CM’s in vivo procoagulant properties. In vitro studies clarified some mechanisms for CM’s procoagulant properties. Thrombin generation assays showed that CM, like skeletal muscle myosin, enhanced thrombin generation in human platelet-rich and platelet-poor plasmas and also in mixtures of purified factors Xa, Va, and prothrombin. Binding studies showed that CM, like skeletal muscle myosin, directly binds factor Xa, supporting the concept that the CM surface is a site for prothrombinase assembly. In tPA (tissue-type plasminogen activator)-induced plasma clot lysis assays, CM was antifibrinolytic due to robust CM-dependent thrombin generation that enhanced activation of TAFI (thrombin activatable fibrinolysis inhibitor). Conclusions: CM in vitro is procoagulant and prothrombotic. CM in vivo can augment myocardial damage and can be prohemostatic in the presence of bleeding. CM’s procoagulant and antifibrinolytic activities likely involve, at least in part, its ability to bind factor Xa and enhance thrombin generation. Future work is needed to clarify CM’s pathophysiology and its mechanistic influences on hemostasis or thrombosis.

Skeletal muscle myosin cross-bridge cycling is necessary for myofibrillogenesis

2003 ◽  
Vol 55 (1) ◽  
pp. 61-72 ◽  
Author(s):  
Indu Ramachandran ◽  
Monica Terry ◽  
Michael B. Ferrari
Keyword(s):  
Skeletal Muscle ◽  
Skeletal Muscle Myosin ◽  
Cross Bridge ◽  
Muscle Myosin
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