Muscle Cross-bridge Cycle Research Articles

Pivoting to a new view of tropomyosin movement.

JGP study (Lehman and Rynkiewicz. 2023. J. Gen. Physiol.https://doi.org/10.1085/jgp.202313387) suggests that tropomyosin regulates the crossbridge cycle in muscle by pivoting around relatively fixed points on actin thin filaments.

Effects of Hydrostatic-Pressure on Muscle Contraction: A Look Back on Some Experimental Findings.

Findings from experiments that used hydrostatic pressure changes to analyse the process of skeletal muscle contraction are re-examined. The force in resting muscle is insensitive to an increase in hydrostatic pressure from 0.1 MPa (atmospheric) to 10 MPa, as also found for force in rubber-like elastic filaments. The force in rigour muscle rises with increased pressure, as shown experimentally for normal elastic fibres (e.g., glass, collagen, keratin, etc.). In submaximal active contractions, high pressure leads to tension potentiation. The force in maximally activated muscle decreases with increased pressure: the extent of this force decrease in maximal active muscle is sensitive to the concentration of products of ATP hydrolysis (Pi-inorganic phosphate and ADP-adenosine diphosphate) in the medium. When the increased hydrostatic pressure is rapidly decreased, the force recovered to the atmospheric level in all cases. Thus, the resting muscle force remained the same: the force in the rigour muscle decreased in one phase and that in active muscle increased in two phases. The rate of rise of active force on rapid pressure release increased with the concentration of Pi in the medium, indicating that it is coupled to the Pi release step in the ATPase-driven crossbridge cycle in muscle. Pressure experiments on intact muscle illustrate possible underlying mechanisms of tension potentiation and causes of muscle fatigue.

The role of myosin loop-4 in localizing tropomyosin on actin during muscle activation and relaxation.

During force-generating steps of the muscle crossbridge cycle, the tip of the myosin motor, namely myosin loop-4, contacts the tropomyosin cable of actin filaments. In the current study, we determined the corresponding effect of myosin loop-4 on the regulatory positioning of tropomyosin on actin. To accomplish this, we compared high-resolution cryo-EM structures of myosin S1-decorated thin filaments containing either wild-type or a loop-4 mutant construct. In the mutant, the seven-residue portion of myosin loop-4 that contacts tropomyosin was replaced by glycine residues, thus removing polar side chains from residues 366 to 372. Cryo-EM analysis of fully-decorated actin-tropomyosin filaments with either wild-type or mutant S1, yielded 3.4- to 3.6-Å resolution reconstructions, with even higher definition at the actin-myosin interface. Loop-4 densities both in wild-type and mutant S1 were clearly identified, and side chains were resolved in the wild-type structure. Aside from loop-4, actin and myosin structural domains were indistinguishable from each other. In marked contrast, the position of tropomyosin on actin in the two reconstructions differed by 3 to 4 Å. In maps of the mutant, tropomyosin was located closer to the myosin-head and thus moved in the direction of the C-state conformation adopted by myosin-free thin filaments. Complementary interaction energy measurements showed that tropomyosin in the mutant thin filaments sits on actin in a local energy minimum site, whereas tropomyosin is positioned by wild-type S1 in an energetically unfavorable location. We propose that the relatively high potential energy associated with wild-type tropomyosin positioning favors an effective transition to B- and C-states following release of myosin from thin filaments during relaxation.

Myosin loop-4 is critical for optimal tropomyosin repositioning on actin during muscle activation and relaxation.

During force-generating steps of the muscle crossbridge cycle, the tip of the myosin motor, specifically loop-4, contacts the tropomyosin cable of actin filaments. In the current study, we determined the corresponding effect of myosin loop-4 on the regulatory positioning of tropomyosin on actin. To accomplish this, we compared high-resolution cryo-EM structures of myosin S1-decorated thin filaments containing either wild-type or a loop-4 mutant construct, where the seven-residue portion of myosin loop-4 that contacts tropomyosin was replaced by glycine residues, thus removing polar side chains from residues 366-372. Cryo-EM analysis of fully decorated actin-tropomyosin filaments with wild-type and mutant S1, yielded 3.4-3.6 Å resolution reconstructions, with even higher definition at the actin-myosin interface. Loop-4 densities both in wild-type and mutant S1 were clearly identified, and side chains were resolved in the wild-type structure. Aside from loop-4, actin and myosin structural domains were indistinguishable from each other when filaments were decorated with either mutant or wild-type S1. In marked contrast, the position of tropomyosin on actin in the two reconstructions differed by 3 to 4 Å. In maps of filaments containing the mutant, tropomyosin was located closer to the myosin-head and thus moved in the direction of the C-state conformation adopted by myosin-free thin filaments. Complementary interaction energy measurements showed that tropomyosin in the mutant thin filaments sits on actin in a local energy minimum, whereas tropomyosin is positioned by wild-type S1 in an energetically unfavorable location. We propose that the high potential energy associated with tropomyosin positioning in wild-type filaments favors an effective transition to B- and C-states following release of myosin from the thin filaments during relaxation.

The Location and Rate of the Phosphate Release Step in the Muscle Cross-Bridge Cycle

It is controversial whether the phosphate (Pi) release step in the cross-bridge cycle occurs before or after the first tension-generating step and whether it is fast or slow. We have therefore modified our previous model of the frog cross-bridge cycle by including a Pi release step either before (model A) or after (model B) the first tension-generating step and refined the two models by downhill simplex runs against experimental data for the force-velocity relation and the tension transients after length steps. Pi release step was initially made slow (70 s−1), but after refinement, it became fast (∼500 s−1 for model A and ∼6000 s−1 for model B). The two models gave similar fits to the experimental tension transients after length steps, but model A gave a better fit to the lengthening limb of the force-velocity relation than model B. 50 mM Pi inhibited the isometric tension of model A by ∼50% but that of model B by only ∼25%. The half-inhibition was at 6.0 mM Pi for model A and at 1.6 mM Pi for model B. The values for model A were consistent with experimental data. We also simulated the effect Pi jump as in caged Pi experiments. For model A, a Pi jump induced a tension fall at a rate similar to the experimental phase II. There was then a small rise in tension to the steady state mimicking the experimental phase III. The initial tension fall was caused by detachment of M⋅ADP⋅Pi myosin heads from actin and reversal of the first tension-generating step. For model B, the fall in tension was more rapid and due to reversal of the first tension-generating step, and phase III was not observed. We conclude that, as in model A, the Pi release step is before the first tension-generating step and is moderately fast.

Reinterpretation of the Tension Response of Muscle to Stretches and Releases

We have reexamined the experimental time courses of tension in frog muscle after rapid length steps. The early tension recoveries are biexponential. After 3 nm/hs stretches and releases, the rates of the immediate rapid tension changes are similar but the subsequent tension fall after a stretch is much slower than the rise after a release. After 1.5 nm/hs length steps, the entire tension responses are more nearly mirror images. To identify the underlying processes, we used a model of the muscle cross-bridge cycle with two tension-generating (tensing) steps. Analysis of the time course of the tension, the rates of the steps in the cycle, and their contributions to tension provided insights into previously puzzling features of the experimental response. After a stretch, the initial rapid tension fall in the model is caused principally by the reversal of the first tensing step, but after a few milliseconds the tensing step resumes its forward direction. We conclude that the remaining response should not be included in phase 2, the period of early tension recovery. With this exclusion, T2, the tension at the end of this period, rises with an increase of stretch. The rate of early tension recovery also increases with stretch size, showing that the reversal of the first tensing step is strain sensitive. After small length steps, the fast and slow components of the early tension recovery are both caused mainly by the first tensing step. The fast component is triggered by the initial sliding of the filaments, and the slow component is due to further sliding that occurs as the tension recovers. With small length steps (<0.5 nm/hs), the time course of the response to a stretch is the reverse of that to a release.

A dynamic approach reveals non-muscle myosin influences the overall smooth muscle cross-bridge cycling rate

Recent evidence suggests that non-muscle myosin IIB (NMIIB) contributes to smooth muscle contraction. This study was designed to determine the effects of NMIIB on the cross-bridge cycling rate. The cross-bridge cycling rate was investigated using sinusoidal analysis. Frequency analysis revealed two asymptotes in the Bode plot of the data; and the intersection of the asymptotes (corner frequency) was higher for the B+/− strain (8.73±1.10Hz vs 16.56±1.26Hz, P<0.05), consistent with a higher overall cross-bridge cycling rate in heterozygous NMIIB KO (B+/−) vs WT mice. These results demonstrate that because of their long duty cycle, NMIIB cross-bridges act as an internal load on smooth muscle myosin to decrease the overall cross-bridge cycling rate and muscle Vmax during force maintenance.

The missing link in the muscle cross-bridge cycle

Four recent studies demonstrate that closing of a cleft in the myosin head is required for actin binding and nucleotide release. This represents a major breakthrough in understanding actomyosin-based motility.

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.

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.

Increased myosin light chain kinase content in sensitized canine saphenous vein.

Our previous studies revealed that smooth muscle from sensitized canine saphenous vein (SCSV) demonstrated greater active shortening capacity, maximum shortening velocity, and prolonged relaxation vis-a-vis the control muscle. These changes could be responsible for the in vivo hyperreactivity of venous smooth muscle observed in anaphylactic shock. Because smooth muscle cross-bridge cycling is regulated by myosin light chain kinase (MLCK)-dependent phosphorylation of the 20-kDa myosin light chain (MLC20), we studied MLC20 and MLCK phosphorylation in homogenates of SCSV and veins from littermate control dogs. We found that phosphorylation of MLC20 in SCSV homogenate was higher (42.26 +/- 5.10%) compared with control homogenates (26.69 +/- 3.30%; P < 0.05); MLCK content was significantly higher in SCSV homogenates [0.169 +/- 0.019 (SE) mu g/mg protein] than in control homogenates (0.075 +/- 0.004 mu g/mg protein; P < 0.05). Total MLCK activity increased from 6.16 +/- 0.60 x 10(-5) nmol Pi x mg fresh weight of tissue-1 x min-1 in control homogenates to 12.50 +/- 2.50 x 10(-5) nmol Pi x mg fresh weight of tissue-1 x min-1 in sensitized homogenates (P < 0.05). Specific MLCK activity was, however, similar in sensitized and control homogenates. The results of our study suggest that elevation of MLCK content in the homogenate could account for the increased contractility of the SCSV in anaphylactic shock.

Effects of Ca2+ on the kinetics of phosphate release in skeletal muscle.

The process of phosphate dissociation during the muscle cross-bridge cycle has been investigated by photoliberation of inorganic phosphate (Pi) within skinned fibers of rabbit psoas muscle. This permitted a test of the idea that Ca2+ controls muscle contraction by regulating the Pi release step of the cycle. Photoliberation of Pi from structurally distinct "caged" Pi precursors initiated a rapid tension decline of up to 12% of active tension, and this was followed by a slower tension decline. The apparent rate constant of the fast phase, kPi, depended on both [Pi] and [Ca2+], whereas the slow phase generally occurred at 2-4 s-1. At maximal Ca2+, kPi increased in a nonlinear manner from 43 +/- 2 s-1 to 118 +/- 7 s-1, as Pi was raised from 0.9 to 12 mM. This was analyzed in terms of a three-state kinetic model in which a force-generating transition is coupled to Pi dissociation from the cross-bridge. As Ca(2+)-activated tension was reduced from maximal (Pmax) to 0.1 Pmax, (i) kPi decreased by up to 2.5-fold, (ii) the relative amplitude of the rapid phase increased 2-fold, and (iii) the relative amplitude of the slow phase increased about 6-fold. Changes in the rapid phase are compatible with Ca2+ influencing an apparent equilibrium constant for the force-generating transition. By comparison, kPi was faster than the rate constant of tension redevelopment, ktr, and was influenced less by Ca2+. Ca2+ effects on the caged Pi transient cannot account for the large effects of Ca2+ on actomyosin ATPase rates or cross-bridge cycling kinetics but may be a manifestation of reciprocal interactions between the thin filament and force-generating cross-bridges, and may represent Ca2+ regulation of the distribution of cross-bridges between non-force-and force-generating states.

Relaxation of muscle fibers with adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) and by laser photolysis of caged ATP[gamma S]: evidence for Ca2+-dependent affinity of rapidly detaching zero-force cross-bridges.

The relationship between the mechanical and biochemical states of the muscle cross-bridge cycle and the control of contraction were investigated by using the nucleotide analogs adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) and caged ATP[gamma S] [the O-1(2-nitrophenyl)ethyl P3-ester of ATP[gamma S]]. ATP[gamma S] interacts with actomyosin in a manner similar to ATP but is hydrolyzed (by a factor of 500) more slowly. Generation of ATP[gamma S] by photolysis of caged ATP[gamma S] within a permeabilized fiber in rigor in the absence of Ca2+ relaxed tension and stiffness as occurs with ATP. The transient rise in tension prior to final relaxation observed with photolysis of caged ATP was absent with caged ATP[gamma S]. This result suggests that following detachment of a cross-bridge, ATP is normally hydrolyzed before force generation. In the presence of Ca2+, photolysis of caged ATP[gamma S] within rigor fibers caused tension to relax fully but significant stiffness remained. Stiffness also developed without concomitant tension when Ca2+ concentration was raised from less than 1 nM to 30 microM in the presence of ATP[gamma S]. The amplitude of the tension response to ramp stretches in the presence of Ca2+ and ATP[gamma S] increased with ramp stretch velocity, suggesting that the cross-bridges have detachment rate constants extending into the 10(3) s-1 range. The results provide evidence that the Ca2+-regulatory system can directly control attachment of cross-bridges into states before the power stroke.

The cross-bridge cycle in muscle. Mechanical, biochemical, and structural studies on single skinned rabbit psoas fibers to characterize cross-bridge kinetics in muscle for correlation with the actomyosin-ATPase in solution.

A characteristic and important feature of myocardium is the modulation of tension when stimulated or possibly even when unstimulated. In addition, resistance to stretch and its variation in unstimulated heart muscle is an important factor in myocardial function. These features may occur in some new light when viewed from some recent advances in understanding of cross-bridge action and regulation of muscle. For this reason we give a short review of such advances. Firstly, we summarize some of our earlier results obtained in experiments designed to see whether and to what extent actomyosin ATPase data obtained in solution might apply in muscle. Secondly, we present a recently developed experimental approach to estimate the rate constants that determine the cycling of cross-bridges between weak-binding, 'non-force-generating' states and strong-binding, 'force-generating' states. The estimated rate constants confirm the prediction of cross-bridge models derived from in vitro studies that the step which is rate-limiting in solution also determines the rate of force-generation in the cross-bridge cycle in muscle. Experiments at various Ca++ concentrations imply that a major mechanism of regulation is the control of the transition from the weak-binding, 'non-force-generating' states to the strong-binding, 'force-generating' states while the number of activated interaction sites appears unchanged and always at its maximum. This implies that changes in the force-pCa relation cannot be interpreted without detailed analysis of cross-bridge kinetics, and that factors other than Ca++ may have the potential to modulate muscle activity, both in stimulated and unstimulated muscle, by affecting cross-bridge kinetics.

Initiation of active contraction by photogeneration of adenosine-5'-triphosphate in rabbit psoas muscle fibres.

Mechanical and biochemical descriptions of the muscle cross-bridge cycle have been correlated. Skinned muscle fibres of rabbit psoas muscle in rigor were incubated in solutions containing approximately equal to 30 microM-Ca2+ ions and P3-1-(2-nitro)phenylethyladenosine-5'-triphosphate, 'caged ATP', an inert photolabile precursor of ATP. ATP was liberated from caged ATP within the fibres by pulses of 347 nm radiation from a frequency-doubled ruby laser. The mechanical responses of muscle fibres to the rapid increase of ATP concentration were monitored. Tension dropped briefly and then rose above the rigor value to the level characteristic of a steady active contraction. Liberation of ATP decreased in-phase stiffness (measured at 500 Hz) from the rigor level to a maintained value intermediate between rigor and relaxed values. Out-of-phase stiffness increased to a maintained level indicating a phase lead of tension with respect to imposed length oscillations. Rigor tension was varied prior to photolysis by slight alterations of fibre length. Tension traces starting at different rigor tensions converged to a common tension level at the same rate, whether or not Ca2+ was included in the medium. These data suggest that the rate of cross-bridge detachment by ATP from the rigor state is not influenced by Ca2+. Analysis of the tension records, in terms of sequential detachment and reattachment reactions, provided a measure of cross-bridge reattachment rate and an alternate measure of the detachment rate. Detachment from the rigor state was approximately proportional to the ATP concentration, with a second-order rate constant of at least 5 X 10(5) M-1 S-1. Reattachment with force generation had no detectable dependence on the concentration of ATP liberated by photolysis. A simple kinetic model of the cross-bridge cycle in terms of chemically defined intermediates was compatible with most of the experimental data. The ATP dependence of cross-bridge detachment, the kinetics of maintained cross-bridge reattachment in the presence of Ca2+, and transient reattachment and final relaxation in the absence of Ca2+ were explained. In this model, reversibility of cross-bridge attachment and the steps leading to force production allow the relatively high observed detachment rate to be accommodated with other data relating to active contraction. These data include the steady ATPase rate of active muscle fibres and the fewer attached cross-bridges in active contractions compared to rigor.