Yin-Biao Sun

The myosin motor in muscle generates a smaller and slower working stroke at higher load

Muscle contraction is driven by the motor protein myosin II, which binds transiently to an actin filament, generates a unitary filament displacement or ‘working stroke’, then detaches and repeats the cycle. The stroke size has been measured previously using isolated myosin II molecules at low load, with rather variable results, but not at the higher loads that the motor works against during muscle contraction. Here we used a novel X-ray-interference technique to measure the working stroke of myosin II at constant load in an intact muscle cell, preserving the native structure and function of the motor. We show that the stroke is smaller and slower at higher load. The stroke size at low load is likely to be set by a structural limit; at higher loads, the motor detaches from actin before reaching this limit. The load dependence of the myosin II stroke is the primary molecular determinant of the mechanical performance and efficiency of skeletal muscle.

Mechanism of force generation by myosin heads in skeletal muscle

Muscles generate force and shortening in a cyclical interaction between the myosin head domains projecting from the myosin filaments and the adjacent actin filaments. Although many features of the dynamic performance of muscle are determined by the rates of attachment and detachment of myosin and actin, the primary event in force generation is thought to be a conformational change or ‘working stroke’ in the actin-bound myosin head. According to this hypothesis, the working stroke is much faster than attachment or detachment, but can be observed directly in the rapid force transients that follow step displacement of the filaments. Although many studies of the mechanism of muscle contraction have been based on this hypothesis, the alternative view—that the fast force transients are caused by fast components of attachment and detachment —has not been excluded definitively. Here we show that measurements of the axial motions of the myosin heads at ångström resolution by a new X-ray interference technique rule out the rapid attachment/detachment hypothesis, and provide compelling support for the working stroke model of force generation.

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 (nH) close to 3, consistent with a single co‐operative transition controlled by Ca2+ binding. Complete inhibition of active force by 25 μm blebbistatin had very little effect on the Ca2+‐dependent structural changes and in particular did not significantly reduce the value of nH. 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 nH 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.

Skeletal muscle resists stretch by rapid binding of the second motor domain of myosin to actin

A shortening muscle is a machine that converts metabolic energy into mechanical work, but, when a muscle is stretched, it acts as a brake, generating a high resistive force at low metabolic cost. The braking action of muscle can be activated with remarkable speed, as when the leg extensor muscles rapidly decelerate the body at the end of a jump. Here we used time-resolved x-ray and mechanical measurements on isolated muscle cells to elucidate the molecular basis of muscle braking and its rapid control. We show that a stretch of only 5 nm between each overlapping set of myosin and actin filaments in a muscle sarcomere is sufficient to double the number of myosin motors attached to actin within a few milliseconds. Each myosin molecule has two motor domains, only one of which is attached to actin during shortening or activation at constant length. A stretch strains the attached motor domain, and we propose that combined steric and mechanical coupling between the two domains promotes attachment of the second motor domain. This mechanism allows skeletal muscle to resist external stretch without increasing the force per motor and provides an answer to the longstanding question of the functional role of the dimeric structure of muscle myosin.

In situ orientations of protein domains: troponin C in skeletal muscle fibers.

A recently developed approach for mapping protein-domain orientations in the cellular environment was used to investigate the Ca(2+)-dependent structural changes in the tropomyosin/troponin complex on the actin filament that regulate muscle contraction. Polarized fluorescence from bifunctional rhodamine probes attached along four alpha helices of troponin C (TnC) was measured in permeabilized skeletal muscle fibers. In relaxed muscle, the N-terminal lobe of TnC is less closed than in crystal structures of the Ca(2+)-free domain, and its D helix is approximately perpendicular to the actin filament. In contrast to crystal structures of isolated TnC, the D and E helices are not collinear. On muscle activation, the N lobe orientation becomes more disordered and the average angle between the C helix and the filament changes by 32 degrees +/- 5 degrees. These results illustrate the potential of in situ measurements of helix and domain orientations for elucidating structure-function relations in native macromolecular complexes.

Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells

Significance Myosin binding protein-C (MyBP-C) is a regulatory protein of heart muscle. Mutations in MyBP-C are frequently associated with heart disease, but the mechanism of action of MyBP-C is poorly understood. By characterizing the effects of its N-terminal domains on the structures of the thin and thick filaments in contracting heart muscle cells, we showed that MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments. The results lead to a model for the control of heart muscle contraction in which the regulatory functions of the thin and thick filaments are coordinated by MyBP-C, providing an integrated framework for the design and development of therapeutic interventions in heart disease. Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.

The molecular basis of the steep force–calcium relation in heart muscle

Contraction of heart muscle is regulated by binding of Ca2+ ions to troponin in the muscle thin filaments, causing a change in filament structure that allows myosin binding and force generation. The steady-state relationship between force and Ca2+ concentration in demembranated ventricular trabeculae is well described by the Hill equation, with parameters EC50, the Ca2+ concentration that gives half the maximum force, and nH, the Hill coefficient describing the steepness of the Ca2+ dependence. Although each troponin molecule has a single regulatory Ca2+ site, nH is typically around 3, indicating co-operativity in the regulatory mechanism. This review focuses on the molecular basis of this co-operativity, and in particular on the popular hypothesis that force-generating myosin cross-bridges are responsible for the effect. Although cross-bridges can switch on thin filaments at low MgATP concentrations, we argue that the evidence from contracting heart muscle cells shows that this mechanism does not operate in more physiological conditions, and would not play a significant role in the intact heart. Interventions that alter maximum force and EC50 do not in general produce a significant change in nH. Complete abolition of force generation by myosin inhibitors does not affect the nH values for either Ca2+ binding to the thin filaments or changes in troponin structure, and both values match that for force generation in the absence of inhibitors. These results provide strong evidence that the co-operative mechanism underlying the high value of nH is not due to force-generating cross-bridges but is rather an intrinsic property of the thin filaments.

Structural changes in troponin in response to Ca2+ and myosin binding to thin filaments during activation of skeletal muscle

Contraction of skeletal and cardiac muscle is regulated by Ca2+-dependent structural changes in troponin that control the interaction between myosin and actin. We measured the orientations of troponin domains in skeletal muscle fibers using polarized fluorescence from bifunctional rhodamine probes on the C and E helices of troponin C. The C helix, in the regulatory head domain, tilts by ≈30° when muscle is activated in physiological conditions, with a Ca2+-sensitivity similar to that of active force. Complete inhibition of active force did not affect C-helix orientation, and binding of rigor myosin heads did not affect its orientation at saturating [Ca2+]. The E helix, in the IT arm of troponin, tilted by ≈10° on activation, and this was reduced to only 3° when active force was inhibited. Binding of rigor myosin heads produced a larger tilt of the E helix. Thus, in situ, the regulatory head acts as a pure Ca2+-sensor, whereas the IT arm is primarily sensitive to myosin head binding. The polarized fluorescence data from active muscle are consistent with an in vitro structure of the troponin core complex in which the D and E helices of troponin C are collinear. The present data were used to orient this structure in the fiber and suggest that the IT arm is at ≈30° to the filament axis in active muscle. In relaxed muscle, the IT arm tilts to ≈40° but the D/E helix linker melts, allowing the regulatory head to tilt through a larger angle.

Effects of sarcomere length and temperature on the rate of ATP utilisation by rabbit psoas muscle fibres

1 The steady state rate of ATP utilisation by single permeabilised fibres from rabbit psoas muscle immersed in silicone oil was measured using a linked enzyme assay that coupled ADP production to the oxidation of NADH. 2 At sarcomere length 2.5 μm, at 10 °C, the rate of ATP utilisation in relaxing conditions was 6 ± 1 μm s−1 (mean ±s.e.m., n= 8 fibres); during isometric contraction it was 310 ± 10 μm s−1 (mean ±s.e.m., n= 11). Assuming a myosin active site concentration of 150 μm, these values correspond to rates of ATP utilisation per active site of about 0.04 and 2.1 s−1, respectively. 3 The rate of ATP utilisation in relaxing conditions was independent of sarcomere length in the range 2.5–4.0 μm. The rate of ATP utilisation during isometric contraction had a dependence on resting sarcomere length similar to that of isometric force in the range 2.5–4.0 μm, but was less strongly dependent on sarcomere length than was isometric force in the range 1.5–2.5 μm. 4 The rate of ATP utilisation in relaxing conditions had a Q10 of 2.5 in the temperature range 7–25 °C, but this increased to 9.7 in the range 25–35 °C, suggesting that some active force may have been generated in relaxing solution at temperatures above 25 °C. 5 The rate of ATP utilisation during isometric contraction had a Q10 of 3.6 throughout the temperature range 7–25 °C; this was similar to the Q10 for isometric force at low temperature (3.5 at 7–10 °C) but much larger than that for isometric force at higher temperature (1.3 at 20–25 °C). 6 Application of the NADH‐linked assay to single muscle fibres in oil improves the effective sensitivity and time resolution of the method, and allows continuous measurements of the rate of ADP production during active contraction.

Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

Significance Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We showed that phosphorylation of the myosin regulatory light chain generates a structural signal that is transmitted between myosin molecules in the thick filament and from the thick to the thin filaments, altering their calcium sensitivity. A closely related dual-filament signaling pathway underlies the enhanced contractility of heart muscle when it is stretched. These coordinated and cooperative changes in thick and thin filament structure are an essential component of contractile regulation in the healthy heart, and their impairment is likely to underlie the functional effects of mutations in thick filament proteins in heart disease. Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease.