Phosphate has dual roles in cross-bridge kinetics in rabbit psoas single myofibrils

In this study, we aimed to study the role of inorganic phosphate (Pi) in the production of oscillatory work and cross-bridge (CB) kinetics of striated muscle. We applied small-amplitude sinusoidal length oscillations to rabbit psoas single myofibrils and muscle fibers, and the resulting force responses were analyzed during maximal Ca2+ activation (pCa 4.65) at 15°C. Three exponential processes, A, B, and C, were identified from the tension transients, which were studied as functions of Pi concentration ([Pi]). In myofibrils, we found that process C, corresponding to phase 2 of step analysis during isometric contraction, is almost a perfect single exponential function compared with skinned fibers, which exhibit distributed rate constants, as described previously. The [Pi] dependence of the apparent rate constants 2πb and 2πc, and that of isometric tension, was studied to characterize the force generation and Pi release steps in the CB cycle, as well as the inhibitory effect of Pi. In contrast to skinned fibers, Pi does not accumulate in the core of myofibrils, allowing sinusoidal analysis to be performed nearly at [Pi] = 0. Process B disappeared as [Pi] approached 0 mM in myofibrils, indicating the significance of the role of Pi rebinding to CBs in the production of oscillatory work (process B). Our results also suggest that Pi competitively inhibits ATP binding to CBs, with an inhibitory dissociation constant of ∼2.6 mM. Finally, we found that the sinusoidal waveform of tension is mostly distorted by second harmonics and that this distortion is closely correlated with production of oscillatory work, indicating that the mechanism of generating force is intrinsically nonlinear. A nonlinear force generation mechanism suggests that the length-dependent intrinsic rate constant is asymmetric upon stretch and release and that there may be a ratchet mechanism involved in the CB cycle.

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Cross-bridge binding to actin and force generation in skinned fibers of the rabbit psoas muscle in the presence of antibody fragments against the N-terminus of actin

Biophysical Journal ◽

10.1016/s0006-3495(96)79579-6 ◽

1996 ◽

Vol 70 (1) ◽

pp. 48-56 ◽

Cited By ~ 2

Author(s):

B. Brenner ◽

T. Kraft ◽

G. DasGupta ◽

E. Reisler

Keyword(s):

Psoas Muscle ◽

Antibody Fragments ◽

Force Generation ◽

Skinned Fibers ◽

Rabbit Psoas ◽

Cross Bridge ◽

N Terminus

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Cross-bridge blocker BTS permits direct measurement of SR Ca2+ pump ATP utilization in toadfish swimbladder muscle fibers

AJP Cell Physiology ◽

10.1152/ajpcell.00025.2003 ◽

2003 ◽

Vol 285 (4) ◽

pp. C781-C787 ◽

Cited By ~ 29

Author(s):

Iain S. Young ◽

Claire L. Harwood ◽

Lawrence C. Rome

Keyword(s):

Sarcoplasmic Reticulum ◽

Direct Measurement ◽

Motor Behavior ◽

Force Generation ◽

Skinned Fibers ◽

Control Value ◽

Cross Bridge ◽

Direct Measurements ◽

Hill Coefficients ◽

Cross Bridges

Because the major processes involved in muscle contraction require rapid utilization of ATP, measurement of ATP utilization can provide important insights into the mechanisms of contraction. It is necessary, however, to differentiate between the contribution made by cross-bridges and that of the sarcoplasmic reticulum (SR) Ca2+ pumps. Specific and potent SR Ca2+ pump blockers have been used in skinned fibers to permit direct measurement of cross-bridge ATP utilization. Up to now, there was no analogous cross-bridge blocker. Recently, N-benzyl- p-toluene sulfonamide (BTS) was found to suppress force generation at micromolar concentrations. We tested whether BTS could be used to block cross-bridge ATP utilization, thereby permitting direct measurement of SR Ca2+ pump ATP utilization in saponin-skinned fibers. At 25 μM, BTS virtually eliminates force and cross-bridge ATP utilization (both <4% of control value). By taking advantage of the toadfish swimbladder muscle's unique right shift in its force-Ca2+ concentration ([Ca2+]) relationship, we measured SR Ca2+ pump ATP utilization in the presence and absence of BTS. At 25 μM, BTS had no effect on SR pump ATP utilization. Hence, we used BTS to make some of the first direct measurements of ATP utilization of intact SR over a physiological range of [Ca2+]at 15°C. Curve fits to SR Ca2+ pump ATP utilization vs. pCa indicate that they have much lower Hill coefficients (1.49) than that describing cross-bridge force generation vs. pCa (∼5). Furthermore, we found that BTS also effectively eliminates force generation in bundles of intact swimbladder muscle, suggesting that it will be an important tool for studying integrated SR function during normal motor behavior.

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Nebulin increases thin filament stiffness and force per cross-bridge in slow-twitch soleus muscle fibers

The Journal of General Physiology ◽

10.1085/jgp.201812104 ◽

2018 ◽

Vol 150 (11) ◽

pp. 1510-1522 ◽

Cited By ~ 8

Author(s):

Masataka Kawai ◽

Tarek S. Karam ◽

Justin Kolb ◽

Li Wang ◽

Henk L. Granzier

Keyword(s):

Soleus Muscle ◽

Thin Filament ◽

Muscle Fibers ◽

Force Generation ◽

Filament Length ◽

Wild Type ◽

Sinusoidal Analysis ◽

Cross Bridge ◽

Significant Difference ◽

Slow Twitch

Nebulin (Neb) is associated with the thin filament in skeletal muscle cells, but its functions are not well understood. For this goal, we study skinned slow-twitch soleus muscle fibers from wild-type (Neb+) and conditional Neb knockout (Neb−) mice. We characterize cross-bridge (CB) kinetics and the elementary steps of the CB cycle by sinusoidal analysis during full Ca2+ activation and observe that Neb increases active tension 1.9-fold, active stiffness 2.7-fold, and rigor stiffness 3.0-fold. The ratio of stiffness during activation and rigor states is 62% in Neb+ fibers and 68% in Neb− fibers. These are approximately proportionate to the number of strongly attached CBs during activation. Because the thin filament length is 15% shorter in Neb− fibers than in Neb+ fibers, the increase in force per CB in the presence of Neb is ∼1.5 fold. The equilibrium constant of the CB detachment step (K2), its rate (k2), and the rate of the reverse force generation step (k−4) are larger in Neb+ fibers than in Neb− fibers. The rates of the force generation step (k4) and the reversal detachment step (k−2) change in the opposite direction. These effects can be explained by Le Chatelier’s principle: Increased CB strain promotes less force-generating state(s) and/or detached state(s). Further, when CB distributions among the six states are calculated, there is no significant difference in the number of strongly attached CBs between fibers with and without Neb. These results demonstrate that Neb increases force per CB. We also confirm that force is generated by isomerization of actomyosin (AM) from the AM.ADP.Pi state (ADP, adenosine diphophate; Pi, phosphate) to the AM*ADP.Pi state, where the same force is maintained after Pi release to result in the AM*ADP state. We propose that Neb changes the actin (and myosin) conformation for better ionic and hydrophobic/stereospecific AM interaction, and that the effect of Neb is similar to that of tropomyosin.

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Differential cross-bridge kinetics of FHC myosin mutations R403Q and R453C in heterozygous mouse myocardium

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01015.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. H91-H99 ◽

Cited By ~ 56

Author(s):

Bradley M. Palmer ◽

David E. Fishbaugher ◽

Joachim P. Schmitt ◽

Yuan Wang ◽

Norman R. Alpert ◽

...

Keyword(s):

Point Mutations ◽

Force Production ◽

Calcium Sensitivity ◽

Isometric Tension ◽

Familial Hypertrophic Cardiomyopathy ◽

Maximal Velocity ◽

In Vitro Motility Assay ◽

Sinusoidal Analysis ◽

Cross Bridge

The kinetic effects of the cardiac myosin point mutations R403Q and R453C, which underlie lethal forms of familial hypertrophic cardiomyopathy (FHC), were assessed using isolated myosin and skinned strips taken from heterozygous (R403Q/+ and R453C/+) male mouse hearts. Compared with wild-type (WT) mice, actin-activated ATPase was increased by 38% in R403Q/+ and reduced by 45% in R453C/+, maximal velocity of regulated thin filament ( VRTF) in the in vitro motility assay was increased by 8% in R403Q/+ and was not different in R453C/+, myosin concentration at half-maximal VRTF was reduced by 30% in R403Q/+ and not different in R453C/+, and the characteristic frequency for oscillatory work production ( b frequency), determined by sinusoidal analysis in the skinned strip at maximal calcium activation, was 27% lower in R403Q/+ and 18% higher in R453C/+. The calcium sensitivity for isometric tension in the skinned strip was not different in R403Q/+ (pCa50 5.64 ± 0.02) and significantly enhanced in R453C/+ (5.82 ± 0.03) compared with WT (5.58 ± 0.02). We conclude that isolated myosin and skinned strips of R403Q/+ and R453C/+ myocardium show marked differences in cross-bridge kinetic parameters and in calcium sensitivity of force production that indicate different functional roles associated with the location of each point mutation at the molecular level.

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BDM affects nucleotide binding and force generation steps of the cross-bridge cycle in rabbit psoas muscle fibers

AJP Cell Physiology ◽

10.1152/ajpcell.1994.266.2.c437 ◽

1994 ◽

Vol 266 (2) ◽

pp. C437-C447 ◽

Cited By ~ 29

Author(s):

Y. Zhao ◽

M. Kawai

Keyword(s):

Equilibrium Constants ◽

Muscle Fibers ◽

Psoas Muscle ◽

Isometric Tension ◽

Activation Mechanism ◽

Phosphate Binding ◽

Rabbit Psoas ◽

Cross Bridge ◽

The Cross ◽

Cross Bridges

The effect of 2,3-butanedione monoxime (BDM) on elementary steps of the cross-bridge cycle was studied with the sinusoidal analysis technique in skinned rabbit psoas muscle fibers. Our results showed that isometric tension and stiffness decreased progressively with an increase in the BDM concentration. The MgATP and MgADP binding constants increased 27 and 6 times, respectively, when BDM was increased from 0 to 18 mM, whereas the phosphate binding constant did not change significantly. The equilibrium constants of the ATP isomerization and detachment step were not sensitive to BDM, whereas the equilibrium constant of the attachment (power stroke) step decreased with BDM. Thus, in the presence of BDM, the number of attached cross bridges decreases; more cross bridges accumulate in the detached state, causing isometric tension and stiffness to decline. However, our detailed analysis shows that the decrease in the number of attached cross bridges is approximately 40%, which is not adequate to account for the 84% decrease in the isometric tension when 18 mM BDM was present. Therefore we suggest that a thin-filament activation mechanism is also affected by BDM.

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Myocardial cross-bridge kinetics in transition to failure in Dahl salt-sensitive rats

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.3.h1390 ◽

2001 ◽

Vol 281 (3) ◽

pp. H1390-H1396 ◽

Cited By ~ 5

Author(s):

Daniel T. McCurdy ◽

Bradley M. Palmer ◽

David W. Maughan ◽

Martin M. LeWinter

Keyword(s):

Mechanical Performance ◽

Dynamic Stiffness ◽

Calcium Sensitivity ◽

Left Ventricular ◽

Isometric Tension ◽

High Salt Diet ◽

Salt Diet ◽

Cross Bridge ◽

Cross Bridges

The role of altered cross-bridge kinetics during the transition from cardiac hypertrophy to failure is poorly defined. We examined this in Dahl salt-sensitive (DS) rats, which develop hypertrophy and failure when fed a high-salt diet (HS). DS rats fed a low-salt diet were controls. Serial echocardiography disclosed compensated hypertrophy at 6 wk of HS, followed by progressive dilatation and impaired function. Mechanical properties of skinned left ventricular papillary muscle strips were analyzed at 6 wk of HS and then during failure (12 wk HS) by applying small amplitude (0.125%) length perturbations over a range of calcium concentrations. No differences in isometric tension-calcium relations or cross-bridge cycling kinetics or mechanical function were found at 6 wk. In contrast, 12 wk HS strips exhibited increased calcium sensitivity of isometric tension, decreased frequency of minimal dynamic stiffness, and a decreased range of frequencies over which cross bridges produce work and power. Thus the transition from hypertrophy to heart failure in DS rats is characterized by major changes in cross-bridge cycling kinetics and mechanical performance.

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Mavacamten has a differential impact on force generation in myofibrils from rabbit psoas and human cardiac muscle

The Journal of General Physiology ◽

10.1085/jgp.202012789 ◽

2021 ◽

Vol 153 (7) ◽

Author(s):

Beatrice Scellini ◽

Nicoletta Piroddi ◽

Marica Dente ◽

Giulia Vitale ◽

Josè Manuel Pioner ◽

...

Keyword(s):

Cardiac Muscle ◽

Striated Muscle ◽

Isometric Force ◽

Inhibitory Effect ◽

Force Generation ◽

Actin Binding ◽

Rabbit Psoas ◽

Force Relaxation ◽

Negative Effect ◽

Sudden Decrease

Mavacamten (MYK-461) is a small-molecule allosteric inhibitor of sarcomeric myosins being used in preclinical/clinical trials for hypertrophic cardiomyopathy treatment. A better understanding of its impact on force generation in intact or skinned striated muscle preparations, especially for human cardiac muscle, has been hindered by diffusional barriers. These limitations have been overcome by mechanical experiments using myofibrils subject to perturbations of the contractile environment by sudden solution changes. Here, we characterize the action of mavacamten in human ventricular myofibrils compared with fast skeletal myofibrils from rabbit psoas. Mavacamten had a fast, fully reversible, and dose-dependent negative effect on maximal Ca2+-activated isometric force at 15°C, which can be explained by a sudden decrease in the number of heads functionally available for interaction with actin. It also decreased the kinetics of force development in fast skeletal myofibrils, while it had no effect in human ventricular myofibrils. For both myofibril types, the effects of mavacamten were independent from phosphate in the low-concentration range. Mavacamten did not alter force relaxation of fast skeletal myofibrils, but it significantly accelerated the relaxation of human ventricular myofibrils. Lastly, mavacamten had no effect on resting tension but inhibited the ADP-stimulated force in the absence of Ca2+. Altogether, these effects outline a motor isoform–specific dependence of the inhibitory effect of mavacamten on force generation, which is mediated by a reduction in the availability of strongly actin-binding heads. Mavacamten may thus alter the interplay between thick and thin filament regulation mechanisms of contraction in association with the widely documented drug effect of stabilizing myosin motor heads into autoinhibited states.

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