Sarcomeric determinants of striated muscle relaxation kinetics

Ca²⁺ is the primary regulator of force generation by cross-bridges in striated muscle activation and relaxation. Relaxation is as necessary as contraction and, while the kinetics of Ca²⁺-induced force development have been investigated extensively, those of force relaxation have been both studied and understood less well. Knowledge of the molecular mechanisms underlying relaxation kinetics is of special importance for understanding diastolic function and dysfunction of the heart. A number of experimental models, from whole muscle organs and intact muscle fibres down to single myofibrils, have been used to explore the cascade of kinetic events leading to mechanical relaxation. By using isolated myofibrils and fast solution switching techniques we can distinguish the sarcomeric mechanisms of relaxation from those of myoplasmic Ca²⁺ removal. There is strong evidence that cross-bridge mechanics and kinetics are major determinants of the time course of striated muscle relaxation whilst thin filament inactivation kinetics and cooperative activation of thin filament by cycling, force-generating cross-bridges do not significantly limit the relaxation rate. Results in myofibrils can be explained well by a simple two-state model of the cross-bridge cycle in which the apparent rate of the force generating transition is modulated by fast, Ca²⁺-dependent equilibration between off- and on-states of actin. Inter-sarcomere dynamics during the final rapid phase of full force relaxation are responsible for deviations from this simple model.     

Kinetics of cardiac sarcomeric processes and rate-limiting steps in contraction and relaxation

The sarcomere is the core structure responsible for active mechanical heart function. It is formed primarily by myosin, actin, and titin filaments. Cyclic interactions occur between the cross-bridges of the myosin filaments and the actin filaments. The forces generated by these cyclic interactions provide the molecular basis for cardiac pressure, while the motion produced by these interactions provides the basis for ejection. The cross-bridge cycle is controlled by upstream mechanisms located in the membrane and by downstream mechanisms inside the sarcomere itself. These downstream mechanisms involve the Ca²⁺-controlled conformational change of the regulatory proteins troponin and tropomyosin and strong cooperative interactions between neighboring troponin-tropomyosin units along the actin filament. The kinetics of upstream and downstream processes have been measured in intact and demembranated myocardial preparations. This review outlines a conceptual model of the timing of these processes during the individual mechanical heart phases. Particular focus is given to kinetic data from studies on contraction-relaxation cycles under mechanical loads. Evidence is discussed that the dynamics of cardiac contraction and relaxation are determined mainly by sarcomeric downstream mechanisms, in particular by the kinetics of the cross-bridge cycle. The rate and extent of ventricular pressure development is essentially subjected to the mechanistic principles of cross-bridge action and its upstream and downstream regulation. Sarcomere relengthening during myocardial relaxation plays a key role in the rapid decay of ventricular pressure and in early diastolic filling.     

Insights into the kinetics of Ca2+-regulated contraction and relaxation from myofibril studies

Muscle contraction results from force-generating interactions between myosin cross-bridges on the thick filament and actin on the thin filament. The force-generating interactions are regulated by Ca²⁺ via specialised proteins of the thin filament. It is controversial how the contractile and regulatory systems dynamically interact to determine the time course of muscle contraction and relaxation. Whereas kinetics of Ca²⁺-induced thin-filament regulation is often investigated with isolated proteins, force kinetics is usually studied in muscle fibres. The gap between studies on isolated proteins and structured fibres is now bridged by recent techniques that analyse the chemical and mechanical kinetics of small components of a muscle fibre, subcellular myofibrils isolated from skeletal and cardiac muscle. Formed of serially arranged repeating units called sarcomeres, myofibrils have a complete fully structured ensemble of contractile and Ca²⁺ regulatory proteins. The small diameter of myofibrils (few micrometres) facilitates analysis of the kinetics of sarcomere contraction and relaxation induced by rapid changes of [ATP] or [Ca²⁺]. Among the processes studied on myofibrils are: (1) the Ca²⁺-regulated switch on/off of the troponin complex, (2) the chemical steps in the cross-bridge adenosine triphosphatase cycle, (3) the mechanics of force generation and (4) the length dynamics of individual sarcomeres. These studies give new insights into the kinetics of thin-filament regulation and of cross-bridge turnover, how cross-bridges transform chemical energy into mechanical work, and suggest that the cross-bridge ensembles of each half-sarcomere cooperate with each other across the half-sarcomere borders. Additionally, we now have a better understanding of muscle relaxation and its impairment in certain muscle diseases.     

Probing S4 and S5 segment proximity in mammalian hyperpolarization-activated HCN channels by disulfide bridging and Cd2+ coordination

We explored the structural basis of voltage sensing in the HCN1 hyperpolarization-activated cyclic nucleotide-gated cation channel by examining the relative orientation of the voltage sensor and pore domains. The opening of channels engineered to contain single cysteine residues at the extracellular ends of the voltage-sensing S4 (V246C) and pore-forming S5 (C303) domains is inhibited by formation of disulfide or cysteine:Cd²⁺ bonds. As Cd²⁺ coordination is promoted by depolarization, the S4–S5 interaction occurs preferentially in the closed state. The failure of oxidation to catalyze dimer formation, as assayed by Western blotting, indicates the V246C:C303 interaction occurs within a subunit. Intriguingly, a similar interaction has been observed in depolarization-activated Shaker voltage-dependent potassium (Kv) channels at depolarized potentials but such an intrasubunit interaction is inconsistent with the X-ray crystal structure of Kv1.2, wherein S4 approaches S5 of an adjacent subunit. These findings suggest channels of opposite voltage-sensing polarity adopt a conserved S4–S5 orientation in the depolarized state that is distinct from that trapped upon crystallization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

桥式交叉组合皮瓣移植修复小腿严重创伤的护理

小腿严重创伤病例在临床越来越多见。当创伤导致主要血管损伤或缺损时，如何有效覆盖创面，保全患者肢体，是摆在医务工作者面前的一大难题。在健肢寻找一组合适的血管，通过桥式交叉的方法为移植组织建立临时血供，当移植组织与受区血液循环建立后再断蒂、分离。而当肢体外伤后需要修复的皮肤软组织面积过于广泛，     

Dependence upon high-energy phosphates of the effects of inorganic phosphate on contractile properties in chemically skinned rat cardiac fibres

The effects of inorganic phosphate (P_i) on mechanical properties of Triton X100 treated ventricular fibres have been studied in different substrate conditions. In the presence of both MgATP and phosphacreatine, increasing concentrations of P_i progressively decreased maximal active force, up to 50–60% at 20 mM P_i. The reduction in stiffness was slightly less. These effects appeared nearly independent of the diameter of the preparations. 20 mM P_i decreased Ca sensitivity of the myofilaments and increased the Hill coefficient of the tension/pCa relationship; furthermore, the time constant of tension recovery was decreased from 12.9 to 8.9 ms suggesting that the cycling rate of cross-bridges was increased in the presence of P_i. When MgATP was regenerated by the myofilament bound creatine kinase in the presence of phosphocreatine, P_i was less efficient in decreasing the maximal tension and it weakened the relaxing effect of MgATP upon rigor tension. These effects are related to the inhibition of creatine kinase by P_i. The effects of P_i on maximal force and kinetics of contraction were antagonized by the effects of a decrease in phosphocreatine. The results are discussed in terms of the antagonistic role of P_i increase and phosphocreatine decrease upon contractile properties of myofilaments during hypoxia in heart muscle.     

Reduction of myocardial cross-bridge turnover rate in presence of EMD 53998, a novel Ca2+-sensitizing agent

We have studied the effect of EMD 53998 (5-(1-(3, 4-dimethoxybenzoyl)-1,2,3,4-tetrahydrochinolin-6-yl)-6-methyl-3, 6-dihydro-2H-1,3,4-thiadiazin-2-one) on cross-bridge turnover rate at varying Ca²⁺ concentrations. Cross-bridge cycling rate was estimated both by adenosine triphosphatase measurements and determination of mechanical characteristics of constantly activated fibres, which is assumed to reflect cross-bridge kinetics. The results indicate that the turnover rate of myocardial cross-bridges was reduced in the presence of EMD 53998 at low Ca²⁺ concentrations (pCa6.25), but not at higher Ca²⁺ concentrations (pCa5.85).     

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

The technique of selective removal of the thin filament by gelsolin in bovine cardiac muscle fibres, and reconstitution of the thin filament from isolated proteins is reviewed, and papers that used reconstituted preparations are discussed. By comparing the results obtained in the absence/presence of regulatory proteins tropomyosin (Tm) and troponin (Tn), it is concluded that the role of Tm and Tn in force generation is not only to expose the binding site of actin to myosin, but also to modify actin for better stereospecific and hydrophobic interaction with myosin. This conclusion is further supported by experiments that used a truncated Tm mutant and the temperature study of reconstituted fibres. The conclusion is consistent with the hypothesis that there are three states in the thin filament: blocked state, closed state, and open state. Tm is the major player to produce these effects, with Tn playing the role of Ca²⁺ sensing and signal transmission mechanism. Experiments that changed the number of negative charges at the N-terminal finger of actin demonstrates that this part of actin is essential to promote the strong interaction between actin and myosin molecules, in addition to the well-known weak interaction that positions the myosin head at the active site of actin prior to force generation.     

Theoretical determination of force-length relations of intact human skeletal muscles using the cross-bridge model

The purpose of this study was to determine force-length relations of selected human skeletal muscles, based on the theoretical foundations of the cross-bridge model and to calculate a strength curve for knee extension from these relations. Force-length relations were determined for the rectus femoris, vastus lateralis, vastus medialis, vastus intermedius and gastrocnemius muscles, using sarcomere/ fiber length data form both legs of four cadavers and sarcomere geometry data reported in the literature. It appears that the two-joint muscles investigated in this study are not able to produce force throughout their full anatomical range of motion, whereas the one-joint muscles can. The strength curve for knee extension was determined as the sum of the force-length relations of the individual knee extensor muscles and showed good agreement with experimentally obtained knee extensor strength curves.     

Mechanical activation and isometric oscillation in insect fibrillar muscle

Summary In isolated contractile structures of insect fibrillar muscle (DLM of Lethocerus) a quick extension causes a delayed rise of tension which is often followed by (damped) isometric oscillations. Similar phenomena may be also observed after a quick release which causes an immediate tension fall followed by a small and nearly immediate tension recovery passing into oscillation. The oscillation frequency has a temperature coefficient (Q ₁₀) of about 3.5 but depends also on the nature of the muscle (8 Hz in DLM of Lethocerus maximus at 20° C; 14 Hz in DLM of Lethocerus annulipes); it is further affected by the ATPase reaction products but not by calcium ions (range 10^–6–10^–5 M) nor by stretch, and it corresponds to the optimum myogenic oscillation frequency in driven oscillation experiments at 0.5% length change as well as to the wingbeat frequency of Lethocerus. All these findings agree with a previous hypothesis (Pringle) that myogenic oscillation — at least at low amplitude and optimal frequency — may be due to a more or less synchronized cross-bridge action. Evidence for partial cross-bridge synchronization during isometric oscillation stems from ATPase estimations in conjunction with measurements of the immediate elasticity (stiffness); these indicate that a maximal number of cross-bridges attaches almost immediately after stretch activation, and nearly in synchrony. The immediate series elasticity determined by quick releases suggests cross-bridge movements of the order 100 Å, in an oscillatory cycle. It is also concluded that contraction speed but not contractile tension is dependent on the turnover frequency of cross-bridges, if, indeed, the latter is reflected in the isometric oscillation frequency.Supported by the Deutsche Forschungsgemeinschaft (Grant RU 154/3-6).