The size and the speed of the working stroke of muscle myosin and its dependence on the force

Myosin II is the motor protein that produces force and shortening in muscle by ATP-driven cyclic interactions of its globular portion, the head, with the actin filament. During each interaction the myosin head undergoes a conformational change, the working stroke, which, depending on the mechanical conditions, can generate a force of several piconewtons or an axial displacement of the actin filament toward the centre of the sarcomere of several nanometres. However, the sizes of the elementary force and length steps and their dependence on the mechanical conditions are still under question. Due to the small fraction of the ATPase cycle time myosin II spends attached to actin, single molecule mechanics failed to produce definitive measurements of the individual events. In intact frog muscle fibres, however, myosin II's working stroke can be synchronised in the few milliseconds following a step reduction in either force or length superimposed on the isometric contraction. Here we show that with 150 μs force steps it is possible to separate the elastic response from the subsequent early rapid component of filament sliding due to the working stroke in the attached myosin heads. In this way we determine how the size and the speed of the working stroke depend on the clamped force. The relation between mechanical energy and force provides a molecular basis for muscle efficiency and an estimate of the isometric force exerted by a myosin head.     

The structural basis of the increase in isometric force production with temperature in frog skeletal muscle

X-ray diffraction patterns were recorded from isolated single fibres of frog skeletal muscle during isometric contraction at temperatures between 0 and 17°C. Isometric force was 43 ± 2% (mean ± s.e.m. , n = 10) higher at 17°C than 0°C. The intensity of the first actin layer line increased by 57 ± 18% ( n = 5), and the ratio of the intensities of the equatorial 1,1 and 1,0 reflections by 20 ± 7% ( n = 10), signalling radial or azimuthal motions of the myosin head domains. The M3 X-ray reflection from the axial repeat of the heads along the filaments was 27 ± 4% more intense at 17°C, suggesting that the heads became more perpendicular to the filaments. The ratio of the intensities of the higher and lower angle peaks of the M3 reflection ( R _M3) was 0.93 ± 0.02 ( n = 5) at 0°C and 0.77 ± 0.02 at 17°C. These peaks are due to interference between the two halves of each myosin filament, and the R _M3 decrease shows that heads move towards the midpoint of the myosin filament at the higher temperature. Calculations based on a crystallographic model of the heads indicated that the observed R _M3 change corresponds to tilting of their light-chain domains by 9 deg, producing an axial displacement of 1.4 nm, which is equal to that required to strain the actin and myosin filaments under the increased force. We conclude that the higher force generated by skeletal muscle at higher temperature can be accounted for by axial tilting of the myosin heads.     

The mechanism of the force response to stretch in human skinned muscle fibres with different myosin isoforms

Force enhancement during lengthening of an active muscle, a condition that normally occurs during locomotion in vivo , is attributed to recruitment of myosin heads that exhibit fast attachment to and detachment from actin in a cycle that does not imply ATP splitting. We investigated the kinetic and mechanical features of this cycle in Ca²⁺ activated single skinned fibres from human skeletal muscles containing different myosin heavy chain (MHC) isoforms, identified with single-fibre gel electrophoresis. Fibres were activated by using a new set-up that allows development of most of the tension following a temperature jump from 0–1°C to the test temperature (∼12°C). In this way we could prevent the development of sarcomere non-uniformity and record sarcomere length changes with a striation follower in any phase of the mechanical protocol. We found that: (i) fibres with fast MHC isoforms develop 40–70% larger isometric forces than those with slow isoforms, as a result of both a larger fraction of force-generating myosin heads and a higher force per head; (ii) in both slow and fast fibres, force enhancement by stretch is due to recruitment of myosin head attachments, without increase in strain per head above the value generated by the isometric heads; and (iii) the extent of recruitment is larger in slow fibres than in fast fibres, so that the steady force and power output elicited by lengthening become similar, indicating that mechanical and kinetic properties of the actin–myosin interactions under stretch become independent of the MHC isoform.     

Temperature change does not affect force between regulated actin filaments and heavy meromyosin in single-molecule experiments

The temperature dependence of sliding velocity, force and the number of cross-bridges was studied on regulated actin filaments (reconstituted thin filaments) when they were placed on heavy meromyosin (HMM) attached to a glass surface. The regulated actin filaments were used because our previous study on muscle fibres demonstrated that the temperature effect was much reduced in the absence of regulatory proteins. A fluorescently labelled thin filament was attached to the gelsolin-coated surface of a polystyrene bead. The bead was trapped by optical tweezers, and HMM–thin filament interaction was performed at 20–35°C to study the temperature dependence of force at the single-molecule level. Our experiments showed that there was a small increase in force with temperature  ( Q ₁₀= 1.43)  and sliding velocity  ( Q ₁₀= 1.46)  . The small increase in force was correlated with the small increase in the number of cross-bridges  ( Q ₁₀= 1.49)  , and when force was divided by the number of cross-bridges, the result did not depend on the temperature  ( Q ₁₀= 1.03)  . These results demonstrate that the force each cross-bridge generates is fixed and independent of temperature. Our additional experiments demonstrate that tropomyosin (Tm) in the presence of troponin (Tn) and Ca²⁺ enhances both force and velocity, and a truncated mutant, Δ23Tm, diminishes force and velocity. These results are consistent with the hypothesis that Tm in the presence of Tn and Ca²⁺ exerts a positive allosteric effect on actin to make actomyosin linkage more secure so that larger forces can be generated.     

Crossbridge and filament compliance in muscle: implications for tension generation and lever arm swing

The stiffness of myosin heads attached to actin is a crucial parameter in determining the kinetics and mechanics of the crossbridge cycle. It has been claimed that the stiffness of myosin heads in the anterior tibialis muscle of the common frog (Rana temporaria) is as high as 3.3 pN/nm, substantially higher than its value in rabbit muscle (~1.7 pN/nm). However, the crossbridge stiffness measurement has a large error since the contribution of crossbridges to half-sarcomere compliance is obtained by subtracting from the half-sarcomere compliance the contributions of the thick and thin filaments, each with a substantial error. Calculation of its value for isometric contraction also depends on the fraction of heads that are attached, for which there is no consensus. Surprisingly, the stiffness of the myosin head from the edible frog, Rana esculenta, determined in the same manner, is only 60% of that in Rana temporaria. In our view it is unlikely that the value of such a crucial parameter could differ so substantially between two frog species. Since the means of the myosin head stiffness in these two species are not significantly different, we suggest that the best estimate of the stiffness of the myosin heads for frog muscle is the average of these data, a value similar to that for rabbit muscle. This would allow both frog and rabbit muscles to operate the same low-cooperativity mechanism for the crossbridge cycle with only one or two tension-generating steps. We review evidence that much of the compliance of the myosin head is located in the pliant region where the lever arm emerges from the converter and propose that tension generation (“tensing”) caused by the rotation and movement of the converter is a separate event from the passive swinging of the lever arm in its working stroke in which the strain energy stored in the pliant region is used to do work.     

Structural changes in myosin motors and filaments during relaxation of skeletal muscle

Structural changes in myosin motors and filaments during relaxation from short tetanic contractions of intact single fibres of frog tibialis anterior muscles at sarcomere length 2.14 μm, 4°C were investigated by X-ray diffraction. Force declined at a steady rate for several hundred milliseconds after the last stimulus, while sarcomere lengths remained almost constant. During this isometric phase of relaxation the intensities of the equatorial and meridional M3 X-ray reflections associated with the radial and axial distributions of myosin motors also recovered at a steady rate towards their resting values, consistent with progressive net detachment of myosin motors from actin filaments. Stiffness measurements confirmed that the fraction of motors attached to actin declined at a constant rate, but also revealed a progressive increase in force per motor. The interference fine structure of the M3 reflection suggested that actin-attached myosin motors are displaced towards the start of their working stroke during isometric relaxation. There was negligible recovery of the intensities of the meridional and layer-line reflections associated with the quasi-helical distribution of myosin motors in resting muscle during isometric relaxation, and the 1.5% increase in the axial periodicity of the myosin filament associated with muscle activation was not reversed. When force had decreased to roughly half its tetanus plateau value, the isometric phase of relaxation abruptly ended, and the ensuing chaotic relaxation had an exponential half-time of ca 60 ms. Recovery of the equatorial X-ray intensities was largely complete during chaotic relaxation, but the other X-ray signals recovered more slowly than force.     

A self-induced translation model of myosin head motion in contracting muscle I. Force-velocity relation and energy liberation

Summary In our previous model, it was assumed that the two heads of myosin act co-operatively in producing force for the sliding of actin filaments relative to myosin filaments. We eliminate the assumption of co-operativity in the present model, following the conclusion by Harada and co-workers that a co-operative interaction between the two heads of myosin is not essential in producing actin filament movement. We assume that (1) a myosin head activated by ATP hydrolysis binds to the thin filament at a definite angle and does not do the power stroke, i.e. does not change its orientation during attachment, (2) a potential of force acting on the myosin head is induced around the thin filament when an ATP-activated myosin head binds to an actin molecule in the thin filament, and (3) the potential remains for a while after detachment of the myosin head and statistically controls the direction of thermal motion of the myosin head, so that the myosin head translates toward the Z-line as a statistical average.We did calculations on these assumptions with a mean tension approximation and got the following results, (a) The calculated force-velocity relation in muscle contraction is in fairly good agreement with experimental observation, including the give phenomenon that lengthening velocity becomes very large for a force about twice the isometric tension. (b) The calculated rate of energy liberation during muscle contraction as a function of load on muscle is in good agreement with experimental results. (c) The calculated distance over which a myosin molecule moves along the thin filament during one ATP hydrolysis can be more than 60 nm under unloaded conditions.     

Nitric oxide plays a role in the regulation of adrenal blood flow and adrenocorticomedullary functions in the llama fetus

The effect of temperature on isometric tension with and without the regulatory proteins tropomyosin and troponin was studied in bovine myocardium using a thin filament removal and reconstitution protocol. In control bovine myocardium, isometric tension increased linearly with temperature in the range 5–40 °C: isometric tension at 10 and 30 °C was 0.65 and 1.28 times that at 20 °C, respectively, with a Q ₁₀ of about 1.4. In actin filament-reconstituted myocardium without regulatory proteins, the temperature effect on isometric tension was less: isometric tension at 10 and 30 °C was 0.96 and 1.17 times that at 20 °C, respectively, with a Q ₁₀ of about 1.1. The temperature dependence of the apparent rate constants was studied using sinusoidal analysis. The temperature dependence of 2π b (rate constant of delayed tension phase) did not vary significantly with the regulatory proteins under the standard activating condition (5 m m MgATP, 8 m m P_i, 200 m m ionic strength, pCa 4.66, pH 7.00). Q ₁₀ for 2π b in control and actin filament-reconstituted myocardium was 3.8 and 4.0, respectively. There were two phases to the temperature dependence of 2π c (rate constant of quick recovery). In control and thin filament-reconstituted myocardium, Q ₁₀ for 2π c was approximately 5.5 in the low temperature range (≤ 25 °C) and 2.7 in the high temperature range (≥ 30 °C). In actin filament-reconstituted myocardium, Q ₁₀ for 2π c was 8.5 in the low temperature range and 3.6 in the high temperature range. The above results demonstrate that regulatory proteins augment the temperature dependence of isometric tension, indicating that the regulatory proteins may modify the actomyosin interaction.     

Crossbridge properties during force enhancement by slow stretching in single intact frog muscle fibres

The mechanism of force enhancement during lengthening was investigated on single frog muscle fibres by using fast stretches to measure the rupture tension of the crossbridge ensemble. Fast stretches were applied to one end of the activated fibre and force responses were measured at the other. Sarcomere length was measured by a striation follower device. Fast stretching induced a linear increase of tension that reached a peak and fell before the end of the stretch indicating that a sudden increase of fibre compliance occurred due to forced crossbridge detachment induced by the fast loading. The peak tension (critical tension, P _c) and the sarcomere length needed to reach P _c (critical length, L _c) were measured at various tensions during the isometric tetanus rise and during force enhancement by slow lengthening. The data showed that P _c was proportional to the tension generated by the fibre under both isometric and slow lengthening conditions. However, for a given tension increase, P _c was 6.5 times greater during isometric than during lengthening conditions. Isometric critical length was 13.04 ± 0.17 nm per half-sarcomere (nm hs⁻¹) independently of tension. During slow lengthening critical length fell as the force enhancement increased. For 90% enhancement, L _c reduced to 8.19 ± 0.039 nm hs⁻¹. Assuming that the rupture force of the individual crossbridge is constant, these data indicate that the increase of crossbridge number during lengthening accounts for only 15.4% of the total force enhancement. The remaining 84.6% is accounted for by the increased mean strain of the crossbridges.     

Calcium- and myosin-dependent changes in troponin structure during activation of heart muscle

Each heartbeat is triggered by a pulse of intracellular calcium ions which bind to troponin on the actin-containing thin filaments of heart muscle cells, initiating a change in filament structure that allows myosin to bind and generate force. We investigated the molecular mechanism of calcium regulation in demembranated trabeculae from rat ventricle using polarized fluorescence from probes on troponin C (TnC). Native TnC was replaced by double-cysteine mutants of human cardiac TnC with bifunctional rhodamine attached along either the C helix, adjacent to the regulatory Ca²⁺-binding site, or the E helix in the IT arm of the troponin complex. Changes in the orientation of both troponin helices had the same steep Ca²⁺ dependence as active force production, with a Hill coefficient ( n _H) close to 3, consistent with a single co-operative transition controlled by Ca²⁺ binding. Complete inhibition of active force by 25 μ m blebbistatin had very little effect on the Ca²⁺-dependent structural changes and in particular did not significantly reduce the value of n _H. Binding of rigor myosin heads to thin filaments following MgATP depletion in the absence of Ca²⁺ also changed the orientation of the C and E helices, and addition of Ca²⁺ in rigor produced further changes characterized by increased Ca²⁺ affinity but with n _H close to 1. These results show that, although myosin binding can switch on thin filaments in rigor conditions, it does not contribute significantly under physiological conditions. The physiological mechanism of co-operative Ca²⁺ regulation of cardiac contractility must therefore be intrinsic to the thin filaments.     

Characterization of the cross-bridge force-generating step using inorganic phosphate and BDM in myofibrils from rabbit skeletal muscles

The inhibitory effects of inorganic phosphate (P_i) on isometric force in striated muscle suggest that in the ATPase reaction P_i release is coupled to force generation. Whether P_i release and the power stroke are synchronous events or force is generated by an isomerization of the quaternary complex of actomyosin and ATPase products (AM.ADP.P_i) prior to the following release of P_i is still controversial. Examination of the dependence of isometric force on [P_i] in rabbit fast (psoas; 5-15 °C) and slow (soleus; 15-20 °C) myofibrils was used to test the two-step hypothesis of force generation and P_i release. Hyperbolic fits of force-[P_i] relations obtained in fast and slow myofibrils at 15 °C produced an apparent asymptote as [P_i]∞ of 0.07 and 0.44 maximal isometric force (i.e. force in the absence of P_i) in psoas and soleus myofibrils, respectively, with an apparent K _d of 4.3 m m in both. In each muscle type, the force-[P_i] relation was independent of temperature. However, 2,3-butanedione 2-monoxime (BDM) decreased the apparent asymptote of force in both muscle types, as expected from its inhibition of the force-generating isomerization. These data lend strong support to models of cross-bridge action in which force is produced by an isomerization of the AM.ADP.P_i complex immediately preceding the P_i release step.     

Influence of ionic strength on the time course of force development and phosphate release by dogfish muscle fibres

We measured the effects of ionic strength (IS), 200 (standard) and 400 mmol l⁻¹ (high), on force and ATP hydrolysis during isometric contractions of permeabilized white fibres from dogfish myotomal muscle at their physiological temperature, 12°C. One goal was to test the validity of our kinetic scheme that accounts for energy release, work production and ATP hydrolysis. Fibres were activated by flash photolysis of the P ³-1-(2 nitrophenyl) ethyl ester of ATP (NPE-caged ATP), and time-resolved phosphate (P_i) release was detected with the fluorescent protein MDCC-PBP, N -(2[1-maleimidyl]ethyl)-7-diethylamino-coumarin-3-carboxamide phosphate binding protein. High IS slowed the transition from rest to contraction, but as the fibres approached the isometric force plateau they showed little IS sensitivity. By 0.5 s of contraction, the force and the rate of P_i release at standard and high IS values were not significantly different. A five-step reaction mechanism was used to account for the observed time courses of force and P_i release in all conditions explored here. Only the rate constants for reactions of ATP, ADP and P_i with the contractile proteins varied with IS, thus suggesting that the actin–myosin interactions are largely non-ionic. Our reaction scheme also fits previous results for intact fibres.     

Reactive oxygen species reduce myofibrillar Ca2+ sensitivity in fatiguing mouse skeletal muscle at 37°C

The mechanisms of muscle fatigue were studied in small muscle bundles and single fibres isolated from the flexor digitorum brevis of the mouse. Fatigue caused by repeated isometric tetani was accelerated at body temperature (37°C) when compared to room temperature (22°C). The membrane-permeant reactive oxygen species (ROS) scavenger, Tiron (5 m m ), had no effect on the rate of fatigue at 22°C but slowed the rate of fatigue at 37°C to that observed at 22°C. Single fibres were microinjected with indo-1 to measure intracellular calcium. In the accelerated fatigue at 37°C the tetanic [Ca²⁺]_i did not change significantly and the decline of maximum Ca²⁺-activated force was similar to that observed at 22°C. The cause of the greater rate of fatigue at 37°C was a large fall in myofibrillar Ca²⁺ sensitivity. In the presence of Tiron, the large fall in Ca²⁺ sensitivity was abolished and the usual decline in tetanic [Ca²⁺]_i was observed. This study confirms the importance of ROS in fatigue at 37°C and shows that the mechanism of action of ROS is a decline in myofibrillar Ca²⁺ sensitivity.     

The elementary force generation process probed by temperature and length perturbations in muscle fibres from the rabbit

Single chemically permeabilized fibres from rabbit psoas muscle were activated maximally at 5–6 °C and then exposed to a rapid temperature increase ('T-jump') up to 37 °C by passing a high-voltage pulse (40 kHz AC, 0.15 ms duration) through the fibre length. Fibre cooling after the T-jump was compensated by applying a warming (40 kHz AC, 200 ms) pulse. Tension and changes in sarcomere length induced by the T-jumps and by fast length step perturbations of the fibres were monitored. In some experiments sarcomere length feedback control was used. After T-jumps tension increased from ∼55 kN m⁻² at 5–6 °C to ∼270 kN m⁻² at 36–37 °C, while stiffness rose by ∼15 %, suggesting that at a higher temperature the myosin head generates more force. The temperature-tension relation became less steep at temperatures above 25°C, but was not saturated even at near-physiological temperature. Comparison of tension transients induced by the T-jump and length steps showed that they are different. The T-jump transients were several times slower than fast partial tension recovery following length steps at low and high temperature (phase 2). The kinetics of the tension rise after the T-jumps was independent of the preceding length changes. When the length steps were applied during the tension rise induced by the T-jump, the observed complex tension transient was simply the sum of two separate responses to the mechanical and temperature perturbations. This demonstrates the absence of interaction between these processes. The data suggest that tension transients induced by the T-jumps and length steps are caused by different processes in myosin cross-bridges.     

Toward a Unified Theory of Muscle Contraction. II: Predictions with the Mean-Field Approximation

The contractile behavior of a single half-sarcomere has been calculated from the lattice model with dimeric myosin and extensible filaments, using the model cycle with two working strokes, explicit Pi-release transitions and faster binding for the second head of the dimer. The mean-field approximation is used to generate independent state probabilities for myosin heads, assuming that the positional symmetry of actin filaments in the half-sarcomere is preserved. This model predicts absolute values of the active tension, stiffness and ATPase of fast fibers and their variation with shortening velocity, the phase-2 tension response to a length-release step and the transient tension rise during ramp stretching, in reasonable agreement with experimental data for frog muscle. It accounts for three observations beyond the reach of traditional models: (i) with elastically stiff myosin, a two-stroke model explains the rate of rapid tension recovery as a function of step size, (ii) slow Pi release from A.M.ADP.Pi after the first stroke generates the flat tension response observed after rapid recovery from a small release step, (iii) a discrete lattice model generates undamped oscillations in the isotonic length response to a force step, as observed when the sarcomeres are highly ordered. The discrete lattice also generates length-dependent oscillations in the tension-length curve and the tension response to ramp shortening, which may be smoothed out if lattice symmetry is broken. An erratum to this article can be found at     

Different effects of cardiac versus skeletal muscle regulatory proteins on in vitro measures of actin filament speed and force

Mammalian cardiac and skeletal muscle express unique isoforms of the thin filament regulatory proteins, troponin (Tn) and tropomyosin (Tm), and the significance of these different isoforms in thin filament regulation has not been clearly identified. Both in vitro and skinned cellular studies investigating the mechanism of thin filament regulation in striated muscle have often used heterogeneous mixtures of Tn, Tm and myosin isoforms, and variability in reported results might be explained by different combinations of these proteins. Here we used in vitro motility and force (microneedle) assays to investigate the influence of cardiac versus skeletal Tn and Tm isoforms on actin–heavy meromyosin (HMM) mechanics. When interacting with skeletal HMM, thin filaments reconstituted with cardiac Tn/Tm or skeletal Tn/Tm exhibited similar speed–calcium relationships and significantly increased maximum speed and force per filament length ( F / l ) at pCa 5 ( versus unregulated actin filaments). However, augmentation of F / l was greater with skeletal regulatory proteins. Reconstitution of thin filaments with the heterogeneous combination of skeletal Tn and cardiac Tm decreased sliding speeds at all [Ca²⁺] relative to thin filaments with skeletal Tn/Tm. Finally, for filaments reconstituted with any heterogeneous mix of Tn and Tm isoforms, force was not potentiated over that of unregulated actin filaments. Combined the results suggest (1) that cardiac regulatory proteins limit the allosteric enhancement of force, and (2) that Tn and Tm isoform homogeneity is important when studying Ca²⁺ regulation of crossbridge binding and kinetics as well as mechanistic differences between cardiac and skeletal muscle.     

Simulation of the rapid regeneration of the actin-myosin working stroke with a tight coupling model of muscle contraction

Summary A. F. Huxley's suggestion in Nature (1992) that a structural modification in the myosin head driven by phosphate release can explain the rapid regeneration of the working stroke, which follows the quick recovery elicited by a step release of moderate size (3–6 nm per half-sarcomere), has been tested with a theoretical model. It is assumed that, in the shortening muscle, cross-bridges can undergo their work producing interaction in two ways distinct for the biochemical state and for the amount of filament sliding allowed. During shortening at low speed, as well as after a shortening step of moderate size, phosphate release from the cross-bridge in the AM-ADP-P state promotes a 100 s^-1 structural change which resets the myosin head in a configuration that allows for a new complete working stroke in the AM-ADP state. In this case the total sliding distance for interaction is about 15 nm. With the increase in shortening velocity a progressively larger fraction of interacting cross-bridges remains in the AM-ADP-P state throughout the working stroke and the sliding distance for interaction is about 11 nm. Reattachment of detached cross-bridges occurs at moderate rate whichever is the pathway from which they originate. The model predicts satisfactorily the time course of the rapid regeneration of the working stroke in double step experiments, but fails to simulate the transition to the steady state response in staircase experiments, the maximum power output during steady shortening and the decrease in rate of energy liberation at high shortening velocities. These results strengthen the conclusion of our previous modelling work where we demonstrated that the condition necessary to fit the mechanical and energetic properties of shortening muscle is to assume two pathways for cross-bridge cycling distinct for the kinetics of detachment and reattachment.     

Ca2+ sensitivity of regulated cardiac thin filament sliding does not depend on myosin isoform

Myosin heavy chain (MHC) isoforms in vertebrate striated muscles are distinguished functionally by differences in chemomechanical kinetics. These kinetic differences may influence the cross-bridge-dependent co-operativity of thin filament Ca²⁺ activation. To determine whether Ca²⁺ sensitivity of unloaded thin filament sliding depends upon MHC isoform kinetics, we performed in vitro motility assays with rabbit skeletal heavy meromyosin (rsHMM) or porcine cardiac myosin (pcMyosin). Regulated thin filaments were reconstituted with recombinant human cardiac troponin (rhcTn) and α-tropomyosin (rhcTm) expressed in Escherichia coli . All three subunits of rhcTn were coexpressed as a functional complex using a novel construct with a glutathione S-transferase (GST) affinity tag at the N-terminus of human cardiac troponin T (hcTnT) and an intervening tobacco etch virus (TEV) protease site that allows purification of rhcTn without denaturation, and removal of the GST tag without proteolysis of rhcTn subunits. Use of this highly purified rhcTn in our motility studies resulted in a clear definition of the regulated motility profile for both fast and slow MHC isoforms. Maximum sliding speed (pCa 5) of regulated thin filaments was roughly fivefold faster with rsHMM compared with pcMyosin, although speed was increased by 1.6- to 1.9-fold for regulated over unregulated actin with both MHC isoforms. The Ca²⁺ sensitivity of regulated thin filament sliding speed was unaffected by MHC isoform. Our motility results suggest that the cellular changes in isoform expression that result in regulation of myosin kinetics can occur independently of changes that influence thin filament Ca²⁺ sensitivity.     

Effects of tropomyosin internal deletion Δ23Tm on isometric tension and the cross-bridge kinetics in bovine myocardium

Tropomyosin (Tm) spans seven actin monomers and contains seven quasi-repeating, loosely similar regions, 1–7. Deletion of regions 2–3 decreases the in vitro sliding speed of synthetic filaments of actin-Tm-Troponin (Tn), and weakens Tm binding to the actin-myosin subfragment 1 (S1) complex (acto-S1). The thin filament was selectively removed from bovine myocardium by gelsolin, and the actin filament was reconstituted, followed by further reconstitution with Tm and Tn. In this reconstitution, full-length Tm (control) was compared with Tm internal deletion mutant Δ23Tm, which lacks residues 47–123 (regions 2–3). The effects of phosphate, MgATP, MgADP and Ca²⁺ were studied in Tm-reconstituted myocardium and Δ23Tm-reconstituted myocardium at pH 7.00 and 25 °C. In Δ23Tm, both isometric tension and stiffness were about 40 % of the control. The Hill factor with Δ23Tm, deduced from the pCa-tension plot, was 1.4 times that of the control, but the Ca²⁺ sensitivity was the same. Sinusoidal analysis indicated that the cross-bridge number in force-generating states was not decreased with Δ23Tm. We conclude that the thin filament cooperativity is increased with Δ23Tm, presumably because of the increased density of the Ca²⁺-binding sites. We further conclude that tension per cross-bridge is 40 % of control and stiffness per cross-bridge is 40 % of control in Δ23Tm. These results are consistent with the idea that Tm modifies the actin-myosin interface so as to increase the stereospecific interaction between moieties of actin and myosin. In Δ23Tm, the interface may not have a perfect stereospecific match so that the tension- and stiffness-generating capacity is greatly diminished.