Masataka Kawai

The elementary step that generates force by cross-bridges (CBs) in striated muscles is reviewed. A literature search focused on models with validating data to verify a CB scheme; models without substantiating data were briefly mentioned or not included. Experimental data include those carried out under the isometric condition in muscle fibers and single myofibrils, along with results from single molecule and stopped-flow studies. These results suggest that force is generated before phosphate (Pi) is released, and the same force is maintained after Pi is released. These studies assumed that Pi is released from myosin. Some results from isotonic experiments are also reviewed, but the data lack the effect of Pi (or a weak effect). Studies with X-ray crystallography and cryo-electron microscopy suggested that force is generated after Pi release from the active site, and Pi is trapped at the secondary site before it is released to the solution. Thus, the difference in the definition of the “Pi release step” must have caused a controversy. It can be concluded that the results from physiological/single molecule studies and cryo-EM/crystal studies complement each other quite well. With isometric experiments, several perturbations are used to generate force transients: length change, chemical change, pressure release, and temperature increase. A small length change includes sinusoidal waveforms, and a large length change includes 10–20% release/restretch. Chemical perturbation includes [Pi] changes. With temperature studies it was shown that the force generation step is endothermic, indicating heat is absorbed. This is qualitatively explained by a hydrophobic interaction between actin and myosin, and by a cleft closure of myosin.

We examined the integrity of flash-frozen and cryo-sectioned cardiac muscle preparations (introduced by Feng and Jin, 2020) by assessing tension transients in response to sinusoidal length changes at varying frequencies (1–100 Hz) at 25 °C. Using 70-μm-thick sections, we isolated fiber preparations to study cross-bridge (CB) kinetics: preparations were activated by saturating Ca²⁺ as well as varying concentrations of ATP and phosphate (Pi). Our results showed that, compared to ordinary skinned fibers, in-series stiffness decreased to 1/2, which resulted in a decrease of isometric tension to 62%, but CB kinetics and Ca²⁺ sensitivity were little affected. The pCa study demonstrated that the rate constant of the force generation step (2πb) is proportionate to [Ca²⁺] at < 5 μM, suggesting that the activation mechanism can be described by a simple second order reaction. We also found that tension, stiffness, and magnitude parameters are related to [Ca²⁺] by the Hill equation, with a cooperativity coefficient of 4–5, which is consistent with the fact that Ca²⁺ activation mechanisms involve cooperative multimolecular interactions. Our results support the long-held hypothesis that Process C (Phase 2) represents the CB detachment step, and Process B (Phase 3) represents the force generation step. Moreover, we discovered that constant H may represent the work-performing step in cardiac preparations. Our experiments demonstrate excellent CB kinetics with two well-defined exponentials that can be more distinguished than those found using ordinary skinned fibers. Flash-frozen and cryo-sectioned preparations are especially suitable for multi-institutional collaborations nationally and internationally because of their ease of transportation.

The elementary molecular step that generates force by cross-bridges (CBs) in active muscles has been under intense investigation in the field of muscle biophysics. It is known that an increase in the phosphate (P_i) concentration diminishes isometric force in active fibers, indicating a tight coupling between the force generation step and the P_i release step. The question asked here is whether the force generation occurs before P_i release or after release. We investigated the effect of P_i on oscillatory work production in single myofibrils and found that P_i-attached state(s) to CBs is essential for its production. Oscillatory work is the mechanism that allows an insect to fly by beating its wings, and it also has been observed in skeletal and cardiac muscle fibers, implying that it is an essential feature of all striated muscle types. With our studies, oscillatory work disappears in the absence of P_i in experiments using myofibrils. This suggests that force is generated during a transition between steps of oscillatory work production, and that the states involved in force production must have P_i attached. With sinusoidal analysis, we obtained the kinetic constants around the P_i release steps, established a CB scheme, and evaluated force generated (and supported) by each CB state. Our results demonstrate that force is generated before P_i is released, and the same force is maintained after P_i is released. Stretch activation and/or delayed tension can also be explained with this CB scheme and forms the basis of force generation and oscillatory work production.

The elementary molecular step that generates force by cross-bridges (CBs) in an active muscle has been under intense investigation in the field of muscle biophysics. The question asked here is whether this step is before phosphate (P_i) release or after its release. It is known that an increase in the concentration of P_i ([P_i]) diminishes isometric force in Ca²⁺ activated fibers, indicating a tight coupling between the force-generating step and the P_i release step. We investigated the effect of P_i on oscillatory work production in single myofibrils and found that P_i-attached state(s) to CBs is essential for its production. Oscillatory work is the mechanism that allows an insect to fly by beating its wings, and it also has been observed in skeletal and cardiac muscle fibers, implying that it is an essential feature of all skeletal muscle types. With our studies, oscillatory work disappears in the absence of P_i in experiments using myofibrils. This suggests that force is generated during a transition between steps of oscillatory work production and that the states involved in its production must have P_i attached. With sinusoidal analysis, we obtained the kinetic constants around the P_i release steps, established a CB scheme, and evaluated force generated (and supported) by each CB state. Our results demonstrate that force is generated before P_i is released, and the same force is maintained after P_i is released. Stretch activation and/or delayed tension can also be explained with this CB scheme and forms the basis of force generation and oscillatory work production.

The use of frozen and cryo-sectioned cardiac muscle preparations, introduced recently by (Feng & Jin, 2020), offers promising advantages of easy transport and exchange of muscle samples among collaborating laboratories. In this report, we examined integrity of such preparation by studying tension transients in response to sinusoidal length changes and following concomitant amplitude and phase shift in tension time courses at varying frequencies. We used sections with 70 µm thickness, isolated fiber preparations, and studied cross-bridge (CB) kinetics: we activated the preparations with saturating Ca²⁺, and varying concentrations of ATP and phosphate (Pi). Our experiments have demonstrated that this preparation has the normal active tension and elementary steps of the CB cycle. Furthermore, we investigated the effect of Ca²⁺ on the rate constants and found that the rate constant r₄ of the force generation step is proportionate to [Ca²⁺] when it is < 5 µM. This observation suggests that the activation mechanism can be described by a simple second order reaction. As expected, we found that magnitude parameters including tension and stiffness are related to [Ca²⁺] by the Hill equation with cooperativity of 4–5, consistent to the fact that Ca²⁺ activation mechanisms involve cooperative multimolecular interactions. Our results are consistent with a long-held hypothesis that process C (phase 2 of step analysis) represents the CB detachment step, and process B (phase 3) represents the force generation step. In this report, we further found that constant H may also represent work performance step. Our experiments have demonstrated excellent CB kinetics with reduced noise and well-defined two exponentials, which are better than skinned fibers, and easier to handle and study than single myofibrils.

The effect of obesity on cross-bridge (CB) function was investigated in mice lacking functional Melanocortin-4 Receptor (MC4R^−/−), the loss of which causes dilated cardiomyopathy (DCM) in humans and mice. Skinned cardiac muscle fibers from male and female mice were used, and activated in the presence of Ca²⁺. To characterize CB kinetics, we changed the length of fibers in sinewaves (15 frequencies: 1‒187 Hz) at a small amplitude (0.2%L₀), studied concomitant tension transients, and deduced the kinetic constants of the CB cycle from the ATP and Pi effects. In males, active tension and stiffness during full activation and rigor were ~ 1.5X in WT compared to MC4R^−/− mice. This effect was not observed in females. We also observed that ATP binding and subsequent CB detachment steps were not altered by the mutation/gender. The equilibrium constant of the force generation step (K₄) and Pi release step (association constant: K₅) were not affected by the mutation, but there was a gender difference in WT mice: K₄ and K₅ were ~ 2.2X in males than in females. Concomitantly, the forward rate constant (r₄) and backward rate constant (r₋₄) of the force generation step were 1.5–2.5X in muscles from female MC4R^−/− mice relative to male MC4R^−/− mice. However, these effects did not cause a significant difference in CB distributions among six CB states. In both genders, Ca²⁺ sensitivity decreased slightly (0.12 pCa unit) in mutants. We conclude that the CB functions are differentially affected both by obesity induced in the absence of functional MC4R^−/− and gender.

We investigated the mechanisms associated with E22K mutation in myosin regulatory light chain (RLC), found to cause hypertrophic cardiomyopathy (HCM) in humans and mice. Specifically, we characterized the mechanical profiles of papillary muscle fibers from transgenic mice expressing human ventricular RLC wild‐type (Tg‐WT) or E22K mutation (Tg‐E22K). Because the two mouse models expressed different amounts of transgene, the B6SJL mouse line (NTg) was used as an additional control. Mechanical experiments were carried out on Ca²⁺‐ and ATP‐activated fibers and in rigor. Sinusoidal analysis was performed to elucidate the effect of E22K on tension and stiffness during activation/rigor, tension‐pCa, and myosin cross‐bridge (CB) kinetics. We found significant reductions in active tension (by 54%) and stiffness (active by 40% and rigor by 54%). A decrease in the Ca²⁺ sensitivity of tension (by ∆pCa ~ 0.1) was observed in Tg‐E22K compared with Tg‐WT fibers. The apparent (=measured) rate constant of exponential process B (2πb: force generation step) was not affected by E22K, but the apparent rate constant of exponential process C (2πc: CB detachment step) was faster in Tg‐E22K compared with Tg‐WT fibers. Both 2πb and 2πc were smaller in NTg than in Tg‐WT fibers, suggesting a kinetic difference between the human and mouse RLC. Our results of E22K‐induced reduction in myofilament stiffness and tension suggest that the main effect of this mutation was to disturb the interaction of RLC with the myosin heavy chain and impose structural abnormalities in the lever arm of myosin CB. When placed in vivo, the E22K mutation is expected to result in reduced contractility and decreased cardiac output whereby leading to HCM.Sub‐Discipline: Bioenergetics.Database: The data that support the findings of this study are available from the corresponding authors upon reasonable request.Animal protocol: BK20150353 (Soochow University).Research governance: School of Nursing: Hua‐Gang Hu: seuboyh@163.com; Soochow University: Chen Ge chge@suda.edu.cn.

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

Vertebrate cardiac muscle generates progressively larger systolic force when the end diastolic chamber volume is increased, a property called the “Frank-Starling Law”, or “length dependent activation (LDA)”. In this mechanism a larger force develops when the sarcomere length (SL) increased, and the overlap between thick and thin filament decreases, indicating increased production of force per unit length of the overlap. To account for this phenomenon at the molecular level, we examined several hypotheses: as the muscle length is increased, (1) lattice spacing decreases, (2) Ca²⁺ sensitivity increases, (3) titin mediated rearrangement of myosin heads to facilitate actomyosin interaction, (4) increased SL activates cross-bridges (CBs) in the super relaxed state, (5) increased series stiffness at longer SL promotes larger elementary force/CB to account for LDA, and (6) stretch activation (SA) observed in insect muscles and LDA in vertebrate muscles may have similar mechanisms. SA is also known as delayed tension or oscillatory work, and universally observed among insect flight muscles, as well as in vertebrate skeletal and cardiac muscles. The sarcomere stiffness observed in relaxed muscles may significantly contributes to the mechanisms of LDA. In vertebrate striated muscles, the sarcomere stiffness is mainly caused by titin, a single filamentary protein spanning from Z-line to M-line and tightly associated with the myosin thick filament. In insect flight muscles, kettin connects Z-line and the thick filament to stabilize the sarcomere structure. In vertebrate cardiac muscles, titin plays a similar role, and may account for LDA and may constitute a molecular mechanism of Frank-Starling response.

The majority of hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere proteins. We examined tropomyosin (Tpm)’s HCM mutants in humans, V95A and D175N, with in vitro motility assay using optical tweezers to evaluate the effects of the Tpm mutations on the actomyosin interaction at the single molecular level. Thin filaments were reconstituted using these Tpm mutants, and their sliding velocity and force were measured at varying Ca2+ concentrations. Our results indicate that the sliding velocity at pCa ≥8.0 was significantly increased in mutants, which is expected to cause a diastolic problem. The velocity that can be activated by Ca2+ decreased significantly in mutants causing a systolic problem. With sliding force, Ca2+ activatable force decreased in V95A and increased in D175N, which may cause a systolic problem. Our results further demonstrate that the duty ratio determined at the steady state of force generation in saturating [Ca2+] decreased in V95A and increased in D175N. The Ca2+ sensitivity and cooperativity were not significantly affected by the mutations. These results suggest that the two mutants modulate molecular processes of the actomyosin interaction differently, but to result in the same pathology known as HCM.