Cross-bridges In Muscle Research Articles

Skeletal muscle stretch and cross-bridge dynamics

Skeletal muscle stretch and cross-bridge dynamics

Rotations of a few cross-bridges in muscle by confocal total internal reflection microscopy

In order to measure the cycling of a few (∼6) myosin heads in contracting skeletal muscle, myofibrils were illuminated by Total Internal Reflection and observed through a confocal aperture. Myosin heads rotated at a rate approximately equal to the ATPase rate, suggesting that bulk ATPase of a whole muscle reflects the cycle frequency of individual heads.

The Effects of Isoflurane on Airway Smooth Muscle Crossbridge Kinetics in Fisher and Lewis Rats

Our aim was to determine how isoflurane modified crossbridge (CB) number and kinetics in airway smooth muscle (ASM) and to compare its effects in Fisher and Lewis rats, two strains with differences in airway responsiveness. The effects of isoflurane (2 MAC) on isotonic and isometric contractility in tracheal ASM strips were investigated after methacholine (10(-6) M)-induced contraction. CB mechanics and kinetics were analyzed using the formalism of Huxley's equations adapted to ASM. After isoflurane, maximum velocity did not differ from baseline in Lewis rats, whereas it was significantly less than baseline in Fisher rats ( approximately 25%), the most reactive strain. Isoflurane totally reversed methacholine-induced increase in active CB number in Lewis rats (2.4 +/- 0.5 versus 1.8 +/- 0.4 10(9)/mm(2) after methacholine and isoflurane, respectively) whereas reversal was only partial in Fisher rats (2.7 +/- 0.4 versus 2.1 +/- 0.3 10(9)/mm(2) after methacholine and isoflurane, respectively). Isoflurane induced a 40% increase in attachment step duration in both strains and an almost twofold increase in the CB cycle duration compared with baseline in Lewis rats. The isoflurane-induced increase in detachment step duration was less in Lewis than in Fisher rats (P < 0.05). We concluded that isoflurane modulated CB number and CB cycling rates of isolated rat ASM differently depending on the level of airway responsiveness.

Quantification and Conformational State of Lysozyme Deposition on Etafilcon, Balafilcon and Lotrafilcon Contact Lens Materials worn on a Daily Wear Basis

Electron paramagnetic resonance spectroscopy was used to monitor the orientation of muscle cross-bridges attached to actin in a low force and high stiffness state that may occur before force generation in the actomyosin cycle of interactions. 2,3-butanedione monoxime (BDM) has been shown to act as an uncompetitive inhibitor of the myosin ATPase that stabilizes a myosin.ADP.P(i) complex. Such a complex is thought to attach to actin at the beginning of the powerstroke. Addition of 25 mM BDM decreases tension by 90%, although stiffness remains high, 40–50% of control, showing that cross-bridges are attached to actin but generate little or no force. Active cross-bridge orientation was monitored via electron paramagnetic resonance spectroscopy of a maleimide spin probe rigidly attached to cys-707 (SH-1) on the myosin head. A new labeling procedure was used that showed improved specificity of labeling. In 25 mM BDM, the probes have an almost isotropic angular distribution, indicating that cross-bridges are highly disordered. We conclude that in the pre-powerstroke state stabilized by BDM, cross-bridges are attached to actin, generating little force, with a large portion of the catalytic domain of the myosin heads disordered.

Smooth muscle myosin: regulation and properties

The relationship of the biochemical states to the mechanical events in contraction of smooth muscle cross-bridges is reviewed. These studies use direct measurements of the kinetics of Pi and ADP release. The rate of release of Pi from thiophosphorylated cycling cross-bridges held isometric was biphasic with turnovers of 1.8 s-1 and 0.3 s-1, reflecting properties and forces directly acting on cross-bridges through mechanisms such as positive strain and inhibition by high-affinity MgADP binding. Fluorescent transients reporting release of an ADP analogue 3'-deac-edaADP were significantly faster in phasic than in tonic smooth muscles. Thiophosphorylation of myosin regulatory light chains (RLCs) increased and positive strain decreased the release rate around twofold. The rates of ADP release from rigor cross-bridges and the steady-state Pi release from cycling isometric cross-bridges are similar, indicating that the ADP-release step or an isomerization preceding it may limit the ATPase rate. Thus ADP release in phasic and tonic smooth muscles is a regulated step with strain- and dephosphorylation-dependence. High affinity of cross-bridges for ADP and slow ADP release prolong the fraction of the duty cycle occupied by strongly bound AM.ADP state(s) and contribute to the high economy of force that is characteristic of smooth muscle. RLC thiophosphorylation led to structural changes in smooth muscle cross-bridges consistent with our findings that thiophosphorylation and strain modulate product release.

The Force Exerted by a Muscle Cross-Bridge Depends Directly on the Strength of the Actomyosin Bond

Myosin produces force in a cyclic interaction, which involves alternate tight binding to actin and to ATP. We have investigated the energetics associated with force production by measuring the force generated by skinned muscle fibers as the strength of the actomyosin bond is changed. We varied the strength of the actomyosin bond by addition of a polymer that promotes protein-protein association or by changing temperature or ionic strength. We estimated the free energy available to generate force by measuring isometric tension, as the free energy of the states that precede the working stroke are lowered with increasing phosphate. We found that the free energy available to generate force and the force per attached cross-bridge at low [Pi] were both proportional to the free energy available from the formation of the actomyosin bond. We conclude that the formation of the actomyosin bond is involved in providing the free energy driving the production of isometric tension and mechanical work. Because the binding of myosin to actin is an endothermic, entropically driven reaction, work must be performed by a “thermal ratchet” in which a thermal fluctuation in Brownian motion is captured by formation of the actomyosin bond.

Skeletal regulatory proteins enhance thin filament sliding speed and force by skeletal HMM

At saturating calcium and nucleotide concentrations, troponin (Tn) and tropomyosin (Tm) enhance the in vitro motility speed of individual actin filaments, suggesting the roles of these thin filament proteins in regulating contraction may include a modulation of crossbridge kinetics. Using a homogeneous complement of fast rabbit skeletal proteins, we examined if Tn and Tm modify specific transitions in the crossbridge cycle by varying skeletal muscle crossbridge kinetics and measuring actin filament sliding speed and steady-state force using the in vitro motility and microneedle assays, respectively. Skeletal regulatory proteins increased the force and sliding speed of actin filaments sliding on skeletal HMM. Faster crossbridge cycling with increased temperature or with substitution of dATP as the contractile substrate resulted in both increased sliding speed and force of unregulated filaments, while the addition of regulatory proteins diminished or eliminated this increase. In contrast, regulatory proteins did not influence filament mechanics when crossbridge cycling was slowed with lowered ATP concentration. The results are most simply explained if addition of the Tn and Tm complex to actin enhances both the transition rate of the force-generating actomyosin isomerization (or the preceding transition) and the apparent crossbridge detachment rate, but that the relative influence of Tn and Tm is dependent on the external load.

Sarcomere-length dependence of lattice volume and radial mass transfer of myosin cross-bridges in rat papillary muscle

We examined the sarcomere length-dependence of the spacing of the hexagonal lattice of the myofilaments and the mass transfer of myosin cross-bridges during contraction of right ventricular papillary muscle of the rat. The lattice spacing and mass transfer were measured by using X-ray diffraction, and the sarcomere length was monitored by laser diffraction at the same time. Although the lattice spacing and the sarcomere length were inversely related, their relationship was not exactly isovolumic. The cell volume decreased by about 15% when the sarcomere length was shortened from 2.3 micro m to 1.8 micro m. Twitch tension increased with sarcomere length (the Frank-Starling law). At the peak tension, the ratio of the intensity of the (1,0) equatorial reflection to that of the (1,1) reflection was smaller when the tension was greater, showing that the larger tension at a longer sarcomere length accompanies a larger amount of mass transfer of cross-bridges from the thick to the thin filament. The result suggests that the Frank-Starling law is due to an increase in the number of myosin heads attached to actin, not in the average force produced by each head.

The Conformation of Myosin Head Domains in Rigor Muscle Determined by X-Ray Interference

In the absence of adenosine triphosphate, the head domains of myosin cross-bridges in muscle bind to actin filaments in a rigor conformation that is expected to mimic that following the working stroke during active contraction. We used x-ray interference between the two head arrays in opposite halves of each myosin filament to determine the rigor head conformation in single fibers from frog skeletal muscle. During isometric contraction (force T 0), the interference effect splits the M3 x-ray reflection from the axial repeat of the heads into two peaks with relative intensity (higher angle/lower angle peak) 0.76. In demembranated fibers in rigor at low force (<0.05 T 0), the relative intensity was 4.0, showing that the center of mass of the heads had moved 4.5 nm closer to the midpoint of the myosin filament. When rigor fibers were stretched, increasing the force to 0.55 T 0, the heads’ center of mass moved back by 1.1–1.6 nm. These motions can be explained by tilting of the light chain domain of the head so that the mean angle between the Cys 707–Lys 843 vector and the filament axis increases by ∼36° between isometric contraction and low-force rigor, and decreases by 7–10° when the rigor fiber is stretched to 0.55 T 0.

Energy storage during stretch of active single fibres.

One of the functions of muscles is to absorb the work done them when they are stretched while active. It has been shown by measurement of ATP hydrolysis that muscle is not a reversible energy conversion device and that work done on a muscle cannot be used to synthesise ATP (Curtin & Davies, 1973). Experiments measuring energy production as heat and work output (Hill & Howarth, 1959; Constable et al 1997) have suggested that work done on an active muscle will eventually become heat but that some of it may be stored in another form before conversion to heat. One way in which this could happen is by storage of work in elastic structures in series with the muscle crossbridges, such as tendon and the muscle filaments themselves. Work would be stored as the stress in these structures rises during a stretch and would be released as the tension falls after the stretch is over. Using measurements of the heat produced by frog single muscle fibres and of the work done on them during stretch we have observed energy storage during stretch, and the release of energy after the end of a stretch. In these preparations the tendon compliance can be kept to very low values, allowing us to investigate whether stretch in series elastic structures can account for the energy stored or whether other structures within the fibre are also capable of energy storage and release.KeywordsEnergy StorageEnergy OutputIsometric ContractionElastic StructureForce EnhancementThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Myosin filament structure and myosin crossbridge dynamics in fish and insect muscles.

The muscle crossbridge power stroke on actin appears to involve a change in angle between the actin-attached motor domain and the neck region of the myosin heads (the ‘tilting neck hypothesis’). However, this mechanism has not been proved beyond doubt and a reasonable question to ask is how actual proof might be achieved. This is essentially a structural question. The question can be put as ‘what are the molecular shapes that the myosin head adopts during the crossbridge cycle on actin?’. A further question that also needs answering is ‘how do the biochemical stages of the actin-myosin ATPase cycle map onto the structural changes that are seen?’. The purpose of the present paper is to address how the first question might be answered and to start to make suggestions about the second.KeywordsFish MuscleMyosin HeadMotor DomainThick FilamentMyosin FilamentThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Polarized Fluorescence Depletion Reports Orientation Distribution and Rotational Dynamics of Muscle Cross-Bridges

The method of polarized fluorescence depletion (PFD) has been applied to enhance the resolution of orientational distributions and dynamics obtained from fluorescence polarization (FP) experiments on ordered systems, particularly in muscle fibers. Previous FP data from single fluorescent probes were limited to the 2 nd- and 4 th-rank order parameters, 〈P 2(cos β)〉 and 〈P 4(cos β)〉, of the probe angular distribution ( β) relative to the fiber axis and 〈P 2d〉, a coefficient describing the extent of rapid probe motions. We applied intense 12- μs polarized photoselection pulses to transiently populate the triplet state of rhodamine probes and measured the polarization of the ground-state depletion using a weak interrogation beam. PFD provides dynamic information describing the extent of motions on the time scale between the fluorescence lifetime (e.g., 4 ns) and the duration of the photoselection pulse and it potentially supplies information about the probe angular distribution corresponding to order parameters above rank 4. Gizzard myosin regulatory light chain (RLC) was labeled with the 6-isomer of iodoacetamidotetramethylrhodamine and exchanged into rabbit psoas muscle fibers. In active contraction, dynamic motions of the RLC on the PFD time scale were intermediate between those observed in relaxation and rigor. The results indicate that previously observed disorder of the light chain region in contraction can be ascribed principally to dynamic motions on the microsecond time scale.

The off rate of Ca(2+) from troponin C is regulated by force-generating cross bridges in skeletal muscle.

The effects of dissociation of force-generating cross bridges on intracellular Ca(2+), pCa-force, and pCa-ATPase relationships were investigated in mouse skeletal muscle. Mechanical length perturbations were used to dissociate force-generating cross bridges in either intact or skinned fibers. In intact muscle, an impulse stretch or release, a continuous length vibration, a nonoverlap stretch, or an unloaded shortening during a twitch caused a transient increase in intracellular Ca(2+) compared with that in isometric controls and resulted in deactivation of the muscle. In skinned fibers, sinusoidal length vibrations shifted pCa-force and pCa-actomyosin ATPase rate relationships to higher Ca(2+) concentrations and caused actomyosin ATPase rate to decrease at submaximal Ca(2+) and increase at maximal Ca(2+) activation. These results suggest that dissociation of force-generating cross bridges during a twitch causes the off rate of Ca(2+) from troponin C to increase (a decrease in the Ca(2+) affinity of troponin C), thus decreasing the Ca(2+) sensitivity and resulting in the deactivation of the muscle. The results also suggest that the Fenn effect only exists at maximal but not submaximal force-activating Ca(2+) concentrations.

Effects of volatile anesthetics on stiffness of mammalian ventricular muscle.

To assess the effects of halothane, isoflurane, and sevoflurane on cross bridges in intact cardiac muscle, electrically stimulated (0.25 Hz, 25 degrees C) right ventricular ferret papillary muscles (n = 14) were subjected to sinusoidal load oscillations (37-182 Hz, 0.2-0.5 mN peak to peak) at the instantaneous self-resonant frequency of the muscle-lever system. At resonance, stiffness is proportional to m * omega(2) (where m is equivalent moving mass and omega is angular frequency). Dynamic stiffness was derived by relating total stiffness to values of passive stiffness at each length during shortening and lengthening. Shortening amplitude and dynamic stiffness were decreased by halothane > isoflurane > or = sevoflurane. At equal peak shortening, dynamic stiffness was higher in halothane or isoflurane in high extracellular Ca(2+) concentration than in control. Halothane and isoflurane increased passive stiffness. The decrease in dynamic stiffness and shortening results in part from direct effects of volatile anesthetics at the level of cross bridges. The increase in passive stiffness caused by halothane and isoflurane may reflect an effect on weakly bound cross bridges and/or an effect on passive elastic elements.

The Smooth Muscle Cross-bridge Cycle Studied Using Sinusoidal Length Perturbations

The mechanical characteristics of smooth muscle can be broadly defined as either phasic, or fast contracting, and tonic, or slow contracting (Somlyo and Somlyo, 1968, Pharmacol. Rev. 20:197–272). To determine if differences in the cross-bridge cycle and/or distribution of the cross-bridge states could contribute to differences in the mechanical properties of smooth muscle, we determined force and stiffness as a function of frequency in Triton-permeabilized strips of rabbit portal vein (phasic) and aorta (tonic). Permeabilized muscle strips were mounted between a piezoelectric length driver and a piezoresistive force transducer. Muscle length was oscillated from 1 to 100 Hz, and the stiffness was determined as a function of frequency from the resulting force response. During calcium activation (pCa 4, 5 mM MgATP), force and stiffness increased to steady-state levels consistent with the attachment of actively cycling cross-bridges. In smooth muscle, because the cross-bridge states involved in force production have yet to be elucidated, the effects of elevation of inorganic phosphate (P i) and MgADP on steady-state force and stiffness were examined. When portal vein strips were transferred from activating solution (pCa 4, 5 mM MgATP) to activating solution with 12 mM P i, force and stiffness decreased proportionally, suggesting that cross-bridge attachment is associated with P i release. For the aorta, elevating P i decreased force more than stiffness, suggesting the existence of an attached, low-force actin-myosin-ADP- P i state. When portal vein strips were transferred from activating solution (pCa 4, 5 mM MgATP) to activating solution with 5 mM MgADP, force remained relatively constant, while stiffness decreased ∼50%. For the aorta, elevating MgADP decreased force and stiffness proportionally, suggesting for tonic smooth muscle that a significant portion of force production is associated with ADP release. These data suggest that in the portal vein, force is produced either concurrently with or after P i release but before MgADP release, whereas in aorta, MgADP release is associated with a portion of the cross-bridge powerstroke. These differences in cross-bridge properties could contribute to the mechanical differences in properties of phasic and tonic smooth muscle.

Superfast contractions without superfast energetics: ATP usage by SR-Ca2+ pumps and crossbridges in toadfish swimbladder muscle.

1. The rate at which an isometrically contracting muscle uses energy is thought to be proportional to its twitch speed. In both slow and fast muscles, however, a constant proportion (25-40 %) of the total energy has been found to be used by SR-Ca2+ pumps and the remainder by crossbridges. We examined whether SR-Ca2+ pumps account for a larger proportion of the energy in the fastest vertebrate muscle known (the toadfish swimbladder), and whether the swimbladder muscle utilizes energy at the superfast rate one would predict from its mechanics. 2. The ATP utilization rates of the SR-Ca2+ pumps and crossbridges were measured using a coupled assay system on fibres skinned with saponin. Surprisingly, despite its superfast twitch speed, the ATP utilization rate of swimbladder was no higher than that of much slower fast-twitch amphibian muscles. 3. The swimbladder achieves tremendous twitch speeds with a modest steady-state ATP utilization rate by employing two mechanisms: having a small number of attached crossbridges and probably utilizing intracellular Ca2+ buffers (parvalbumin) to spread out the time over which Ca2+ pumping can occur. 4. Finally, although the total ATP utilization rate was not as rapid as expected, the relative proportions used by SR-Ca2+ pumps and the crossbridges were similar to other muscles.

The ADP Release Step of the Smooth Muscle Cross-Bridge Cycle Is Not Directly Associated with Force Generation

When smooth muscle myosin subfragment 1 (S1) is bound to actin filaments in vitro, the light chain domain tilts upon release of MgADP, producing a ∼3.5-nm axial motion of the head-rod junction (Whittaker et al., 1995. Nature. 378:748–751). If this motion contributes significantly to the power stroke, rigor tension of smooth muscle should decrease substantially in response to cross-bridge binding of MgADP. To test this prediction, we monitored mechanical properties of permeabilized strips of chicken gizzard muscle in rigor and in the presence of MgADP. For comparison, we also tested psoas and soleus muscle fibers. Any residual bound ADP was minimized by incubation in Mg2+-free rigor solution containing 15mM EDTA. The addition of 2mM MgADP, while keeping ionic strength and free Mg2+ concentration constant, resulted in a slight increase in rigor tension in both gizzard and soleus muscles, but a decrease in psoas muscle. In-phase stiffness monitored during small (<0.1%) 500-Hz sinusoidal length oscillations decreased in all three muscle types when MgADP was added. The changes in force and stiffness with the addition of MgADP were similar at ionic strengths from 50 to 200mM and were reversible. The results with gizzard muscle were similar after thiophosphorylation of the regulatory light chain of myosin. These results suggest that the axial motion of smooth muscle S1 bound to actin, upon dissociation of MgADP, is not associated with force generation. The difference between the present mechanical data and previous structural studies of smooth S1 may be explained if geometrical constraints of the intact contractile filament array alter the motions of the myosin heads.

Viscous properties of human muscle during contraction

The purpose of this study was to determine viscous properties of human muscle during plantarflexion efforts. Experiments were performed on 17 subjects with an ankle ergometer allowing sinusoidal oscillations during isometric contractions and isokinetic movements. Sinusoidal oscillations led to the expression of (i) Bode diagrams of the musculo-articular system allowing the determination of a damping coefficient ( B bode); and (ii) a viscous coefficient ( B sin) using an adaptation of Hill’s equation to sinusoidal oscillations. Isokinetic movements led to torque–velocity relationships. They showed a fall in torque associated to an increase in angular velocity what was quantified by calculating a damping coefficient ( B iso). Both experiments gave consistent results indicating that B bode was the lowest viscous parameter. This difference is discussed in terms of (i) “analog” viscosity originating from muscle cross-bridges; and (ii) real mechanical damping of passive structures.

Troponin C regulates the rate constant for the dissociation of force-generating myosin cross-bridges in cardiac muscle.

It is well known that cardiac troponin C (cTnC) regulates the association of force-generating myosin cross-bridges. We report here evidence for an additional role for cTnC. This hypothesis states that Ca2+ binds more strongly to cTnC when force-generating myosin cross-bridges are attached to actin and that removal of this bound Ca2+ accelerates the dissociation of force-generating myosin cross-bridges. Intact Fura-2-loaded rat papillary muscles and skinned (permeabilized) ventricular preparations were used. The preparations were mounted in the Guth Muscle Research System which is capable of measuring simultaneously fluorescence and force in response to length perturbations. All mechanical perturbations of muscle length (isotonic shortening, quick stretches and releases, and length vibrations) which cause dissociation of force-generating myosin cross-bridges during a twitch resulted in Ca2+ being released from troponin as judged from changes in the Ca2+ transients (Fura-2 (340/380) fluorescence ratio). Thus dissociation of force-generating myosin cross-bridges cause Ca2+ to be released from cTnC. Conversely, it would be expected that removal of strongly bound Ca2+ from cTnC would result in an increase in the rate of dissociation of force-generating myosin cross-bridges. To test this hypothesis actomyosin ATPase (NADH fluorescence change) and isometric force were measured in skinned cardiac preparations. The ratio of the ATPase/Force is proportional to the rate constant (gapp) for the dissociation of force-generating myosin cross-bridges. The data showed that decreasing the amount of Ca2+ bound to cTnC in skinned cardiac fibers caused an increase in the ratio of ATPase/Force, the rate of dissociation (gapp) of force-generating myosin cross-bridges.

Regulation of the cross-bridge cycle: the effects of MgADP, LC17 isoforms and telokin.

This review summarizes the role of MgADP in force maintenance by dephosphorylated cross-bridges in smooth muscle and a potential physiological role for telokin. In tonic, compared with phasic, smooth muscles the affinity of cross-bridges in approximately 5 times higher for MgADP and the apparent second-order rate constant for MgATP is approximately 3 times lower. This gives rise to a large population of dephosphorylated cross-bridges in tonic smooth muscle. Such cross-bridges are thought to be major determinants of the different relaxation kinetics of the two types of smooth muscle and contribute to force maintenance at low levels of MLC20 phosphorylation, termed 'catch-like state' (Somlyo & Somlyo 1967) or 'latch' (Dillon et al. 1981). The molecular basis of the different affinities for MgADP and MgATP between tonic and phasic smooth muscle myosin was explored by exchange of essential myosin light chain (LC17) isoforms. In phasic bladder smooth muscle the exchange of LC17b for LC17a caused a significant decrease in the unloaded shortening velocity of non-phosphorylated, slowly cycling cross-bridges, suggesting that the LC17 isoforms contribute to the nucleotide affinity of latch bridges. The role of telokin in Ca(2+)-desensitization in phasic smooth muscle is reviewed. Telokin, the independently expressed C-terminus of myosin light chain kinase, is extensively phosphorylated during forskolin- and 8-br-cGMP-induced relaxation in situ. Telokin accelerated dephosphorylation of the regulatory myosin light chain and relaxed rabbit ileum smooth muscle. The results suggest that telokin contributes to cAMP and/or cGMP kinase-mediated Ca(2+)-desensitization of phasic smooth muscles.