Relaxation of chemically skinned guinea pig taenia coli smooth muscle from rigor by photolytic release of adenosine-5′-triphosphate

Summary The mechanical events following release of ATP from P³-1-(2-nitro)phenylethyladenosine-5-triphosphate (caged-ATP) in skinned guinea pig taenia coli smooth muscle in rigor were investigated. A rigor force of about 25–35% of the maximal active force was obtained by removing ATP at the plateau of a maximal active contraction. In the rigor solution free-Mg²⁺ was 2mm, ionic strength 90mm and pH 7.0. When caged-ATP (12.5mm) was diffused into the preparation there was no change in the rigor force. Photolytic production of about 2mm ATP was achieved with a xenon flash lamp. Following illumination, force decreased with an approximate initial rate constant of 0.7 s^–1. The rate of relaxation was increased in the presence of inorganic phosphate (at 3mm: 1.3 s^–1; 10mm: 2.2 s^–1). At higher Mg²⁺ concentrations the rate of relaxation was slower (5mm: 0.2 s^–1) and at lower concentrations the rate was faster (0.5mm: 1.2 s^–1). An increased rate of relaxation was observed when ionic strength was increased to 150mm (2.2 s^–1). Phosphate increased the rate of relaxation at the different levels of Mg²⁺ (0.5–10mm) and ionic strength (90, 150mm). In preparations shortened (by 1–3%) to give reduced rigor force, a small transient increase in tension was recorded after ATP release. In comparison to the rates of ATP-induced dissociation of actomyosin in solution, reported in the literature, the rate of relaxation from rigor is slower. This may reflect a slow rigor cross-bridge dissociation or mechanical interactions not associated with cross-bridges in the muscle fibre. However, the results may also be interpreted on the basis of a model proposed for striated muscle by Goldmanet al. (1984) where the relaxation from rigor in the absence of Ca²⁺ involves a phase of reattaching cross-bridges whose lifetime in a tension-producing state is influenced by phosphate.     

Series elastic properties of skinned muscle fibres in contraction and rigor

Summary Isometric tension of skinned fibres from the frog semitendinosus muscle is sigmoidally related to Ca²⁺ concentration betweenpCa 7 and 6. Stiffness measurements showed that the Ca²⁺-activated tension may be due to recruitment of attached cross-bridges. In the absence of ATP (rigor solution) the skinned fibre develops a rigor tension which reaches about 80–110% of the maximum Ca²⁺-activated tension.However, stiffness measurements showed that in rigor many more cross-bridges are attached to actin at any one moment than in contraction. It was concluded that the force per cross-bridge is 37% smaller in rigor than in contraction.     

Temperature sensitivity of force and shortening velocity in maximally activated skinned smooth muscle

We have studied the temperature dependence of isometric force, rate of force development and maximal shortening velocity (V _max) in skinned guinea-pig taenia coli smooth muscle. To eliminate the influence of temperature on activation mechanisms, maximally thiophosphorylated preparations were used. Isometric force in the range 2–35°C was maximal at 22°C with a decrease of 25% at 2°C and 10% at 35°C. Rate of tension development from rigor after photolytic release of ATP increased four-fold between 5°C and 30°C. V _max increased with a Q₁₀ of about 2 (1.6, range 5–15°C, and 2.2, range 22–30°C). The temperature dependence of the rate of tension development indicates rate-limitation by transitions into force-generating states or by the hydrolysis reaction. The temperature dependence of V _max reflects effects of temperature on reactions (e.g. the ADP-release) associated with cross-bridge detachment. The small temperature dependence of steady-state force in smooth compared with skeletal muscle suggests differences in the cross-bridge reactions controlling the number of attached force-generating states in the two muscle types. This revised version was published online in August 2006 with corrections to the Cover Date.     

The influence of ionic strength upon relaxation from rigor induced by flash photolysis of caged-ATP in skinned murine skeletal muscle fibres

The influence of ionic strength upon relaxation kinetics from rigor in skinned murine extensor digitorum longus (EDL) skeletal muscle fibres was examined using photolysis of caged-ATP at low Ca²⁺. The ionic strength was adjusted with either KMeSO₃ or ethylene glycol bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid, dipotassium salt (K₂EGTA) in the range of /2=65–215 mM, or I.E. 49–194 mM, where I.E. denotes ionic equivalent. Following rigor development at a/2 of 165–215 mM (I.E. 144–194 mM), the liberation of approximately 0.5 mM ATP resulted in an initial 6-to 10-ms detachment phase with a decline in force of approximately 10–20% followed by a 10-to 30-ms reattachment with up to a 60% increase compared to the corresponding rigor level and a final detachment leading to complete relaxation. Interestingly, when similar ATP concentrations were liberated at lower ionic strengths between a /2 of 65 mM and 110 mM (I.E. 60–100 mM), the initial detachment phase was shortened and force decreased by only approximately 5–10%, while the following reattachment phase was lengthened and led to an increased steady-state force of approximately 20–80% without final relaxation. ATP-induced detachment and subsequent reattachment were mainly determined by the currently present ionic strength and were relatively independent of the preceding rigor state which had been developed at higher or lower ionic strengths. The effects of phosphate and apyrase on the force transient suggest that reattachment of ADP- binding crossbridges may contribute to the increase in tension at high and even more at low ionic strengths. The study shows that the kinetics of initial fast relaxation and subsequent redevelopment of force following flash photolysis of similar ATP concentrations are markedly modified by the ionic strength in the narrow range of between 65 mM and 215 mM.     

What does TnCDANZ fluorescence reveal about the thin filament state?

Fluorescence of skinned psoas fibres reconstituted with the troponin C subunit labelled with the fluorescent probe dansylaziridine (TnC_DANZ) increases upon activation with Ca²⁺. This fluorescence enhancement is due to Ca²⁺ binding to the Ca²⁺-specific binding sites of TnC_DANZ and attachment of cross-bridges to the actin filament. We found that approximately 20% of the enhanced fluorescence signal derived from Ca²⁺ binding to TnC_DANZ and 80% from cross-bridge attachment during maximal activation. Furthermore we studied the effects of different cross-bridge states on TnC_DANZ fluorescence. Weakly bound, non-force-generating cross-bridge states (pCa 8, low ionic strength) and rigor cross-bridges revealed similar effects on the TnC_DANZ fluorescence. Strongly attached, force-generating states, however, increased fluorescence to the greatest extent. These results suggests a complex system of reciprocal couplings between TnC and different attached cross-bridge states. Cooling or increase of inorganic phosphate decreased isometric force but hardly decreased fluorescence, suggesting the accumulation of attached cross-bridge states with low tension output.     

Rigor contraction and the effect of various phosphate compounds on glycerinated insect flight and vertebrate muscle

1. Glycerol-extracted flight muscle from the giant water-bug, Lethocerus cordofanus, undergoes contraction when deprived of ATP. This rigor contraction occurs in the presence of the calcium chelating agent EGTA, under conditions in which the predicted free calcium ion concentration is less than 5 × 10^-9 M. A mechanically similar contraction has been observed by depriving the muscle of Mg²⁺, in the presence of 5 mM-ATP.2. In the rigor contraction tensions of up to 120 mN/mm² under isometric conditions, and shortenings of between 2½ and 6% under isotonic conditions, have been observed at 20° C.3. Muscles in rigor can be relaxed by the addition of ATP. The minimum concentration of ATP required to give full relaxation depends upon other ionic constituents of the solutions, and upon temperature, but is between 0·3 and 1 mM at 20° C. Lower concentrations result in partial reduction of tension and stiffness.4. Pyrophosphate (PP) causes a reduction in the tension of muscle which has developed rigor under isometric conditions, but the stiffness, when measured at frequencies between 1 and 100 Hz, remains indistinguishable from that of rigor muscle. The stiffness when measured by applying slow length changes is comparable to that of ATP-relaxed muscle. It is suggested that in PP-relaxed muscle the cross-bridges remain in close proximity to, but are not rigidly attached to, the I filaments, resulting in a high viscous interaction between the two sets of filaments.5. The addition of low concentrations of ADP (less than about 0·7 mM) to rigor muscle in the absence of ATP causes an increase in tension. The effect is sigmoid, and is probably due to the formation of low concentrations of ATP throughout the body of the fibre by myokinase activity. Larger concentrations of ADP (about 5 mM) added to rigor muscle cause relaxation, probably due to the formation of higher ATP concentrations.6. AMP and inorganic orthophosphate have little effect upon the mechanical properties of rigor muscle.7. There is a delay of about 1 min before the onset of rigor contraction when fibres are transferred from an ATP-solution to one containing no ATP, due to the transfer of ATP in the body of the fibres. Both ATP hydrolysis by the fibres and diffusion of ATP into the bathing solution contribute significantly to the rate of depletion of ATP.8. Rabbit psoas muscle shows a similar rigor contraction in the presence of EGTA, and has mechanical properties similar to those described for Lethocerus flight muscle in the presence of pyrophosphate.     

Generation of tension by glycerol-extracted vertebrate skeletal muscle fibres in the absence of calcium

Summary When a small bundle of glycerol-extracted fibres from either frog, tortoise or rabbit skeletal muscle was first exposed to high MgATP (5mm) in the absence of Ca²⁺ (<1nm) and at low ionic strength (<0.11) at 20° C, it produced a single sharp transient contraction followed by a lower maintained isometric tension. The maintained tension was investigated further in rabbit psoas fibres. Ca²⁺-free tension was dependent on the ionic strength in the range 0.04–0.10, on the temperature in the range 6–20° C and the free Mg²⁺ in the range 0–6mm. It was promoted by low ionic strength, low Mg²⁺ and high temperature, and was unaffected by varying the MgATP^2– in the range 0.4–4mm and by adding ATP regenerating components. A separate regime of tension generation was detected at MgATP^2– concentrations of less than 0.1mm, in which MgATP^2– concentration was critical. The results are interpreted on the assumption that binding of Mg²⁺ to some component of the regulatory system is necessary to maintain its inhibitory effect in the absence of Ca²⁺. Ionic strength and temperature, on the other hand, may affect actomyosin directly.     

Mechanical characteristics of skinned and intact muscle fibres from the giant barnacle,Balanus nubilus

Intact muscle fibres fromBalanus nubilus develop tensions of up to 600 kN sd m⁻² during electrical stimulation. The rise of tension occurs with a half-time (177 ms at 12° C) about fivefold longer than that of tetanised frog muscle at the same temperature. The response of myofibrillar bundles to a rapid stretch resembles that of frog muscle but has a y_o value (i.e. the size of an instantaneous release necessary to just discharge tension) which is ca. 2.5 times smaller, and phase 2 of the tension transient (the “quick phase”) occurs at a rate comparable to that of frog muscle. In contrast, the ATPase activity (0.018 mmoles · kg wet weight⁻¹ · s⁻¹) of this preparation and its maximum shortening velocity (0.15–0.16 muscle lengths · s⁻¹) are both at least fivefold slower than frog muscle. These findings can be accounted for by a cross-bridge cycle in barnacle muscle in which events prior and subsequent to the tension generating step(s) occur at a rate at least fivefold slower than comparable steps in frog muscle, but the step(s) associated with tension development occur at similar rates in the two preparations. Since the rate of mechanical relaxation in barnacle muscle is modified in the presence of intracellular calcium buffers and by depolarisation-induced elevation of the free calcium during the relaxation phase, it is proposed that the time course of relaxation is not determined exclusively by the kinetics of the cross-bridge cycle, but is also dependent on the free calcium concentration during relaxation.     

Activation of skinned muscle fibres from the Norway lobster Nephrops norvegicus L. by manganese ions

Effects of Mn2+ and Ca2+ on the mechanical properties of glycerinated myofibrillar bundles originating from slow S1 type muscle fibres of superficial flexor muscles of the lobster Nephrops norvegicus were investigated. Mn2+ (5–20μm) activated the preparations in a dose-dependent manner. The sensitivity of myofibrillar force generation for Mn2+ was around 30 times lower than that for Ca2+. The maximal tension produced under Mn2+ activation was about 75% of that under Ca2+ activation. At higher free Mn2+ concentrations (>2mm), the steady-state force decreased; it was completely abolished at 30mm free Mn2+. These high Mn2+ solutions were accompanied by changes in MgATP and MnATP concentrations, and in the ionic strength. Control experiments have shown that none of these parameters seemed fo account fully for the observed force depression in high Mn2+ solutions. It is likely that direct effects of Mn2+ such as a change of the myofilament surface charges are responsible. The maximal unloaded shortening velocity of the myofibrillar preparations was shown to be similar under maximal Mn2+ and Ca2+ activation. Conversely, the kinetics of stretch-induced delayed force increase were about two to three times faster under Mn2+ activation. These results suggest that certain steps of the cross-bridge cycle depend on the ion species bound to the regulatory proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of ionic strength on crossbridge kinetics as studied by sinusoidal analysis,ATP hydrolysis rate and x-ray diffraction techniques in chemically skinned rabbit psoas fibres

Summary In order to identify which steps in the crossbridge cycle are affected by changes in ionic strength, we studied the effect of ionic strength on the rate constants and magnitudes of three exponential processes, the ATP hydrolysis rate and isometric tension during maximal activation (pCa 4.52, 5 mM MgATP). Equatorial X-ray diffraction measurements were also carried out in both relaxing and rigor conditions to examine whether the distance between thick and thin filaments changes with ionic strength (range: 100–300 mM). All experiments were carried out at 20° C and at pH 7.0 on chemically skinned rabbit psoas muscle fibres. Isometric tension and muscle stiffness declined significantly as the ionic strength was increased from 150 mM to 300 mM. The concomitant decrease in the ATP hydrolysis rate was much less than tension, resulting in a large increase in the tension cost. Three rate constants of exponential processes, deduced from sinusoidal analysis, did not change appreciably. The magnitude parameters of all three processes diminished as the ionic strength was increased. During relaxation the filament spacing increased by 5% when the ionic strength was increased from 150 mM to 300 mM. After rigor induction, the spacing did not change with ionic strength. We conclude that a change in ionic strength modifies the rapid equilibrium between the detached state and the weakly attached state, and that this causes considerable effect on isometric tension. We also conclude that other steps in the crossbridge cycle are less sensitive to ionic strength, and that the lattice spacing change is unable to account for the considerable effect of ionic strength on isometric tension.     

Effects of 2,3-butanedione monoxime on cross-bridge kinetics in rat cardiac muscle

The effects of 2,3-butanedione monoxime (BDM) on isometric force and myofibrillar adenosine 5′-triphosphatase (ATPase) activity were studied in skinned cardiac trabeculae from the rat. ATP hydrolysis was enzymatically coupled to the breakdown of reduced nicotinamide adeninedinucleotide (NADH). The NADH concentration was monitored photometrically. Measurements were performed at a sarcomere length of 2.1 μm, 20 °C and pH 7.0. Without BDM, isometric force was 45 ± 3 kN/m² and the isometric ATPase activity 0.49 ± 0.04 mM/s (mean ± SEM, n = 31). Force gradually decreased as a function of [BDM] to 2.8 ± 0.4% at 100 mM BDM. ATPase activity was also depressed by BDM, but to a lesser extent than force. BDM therefore has a marked effect on myofibrillar tension cost. The rate of tension redevelopment after unloaded shortening decreased from 29 ± 2 s⁻¹ (n = 10) without BDM to 22 ± 1 s⁻¹ (n = 5) at 20 mM BDM. These results, modelled in a two- and three-state scheme of cross-bridge interaction, indicate that, in cardiac muscle, BDM not only affects cross-bridge formation but, especially at high concentrations (≥ 20 mM), also causes a marked increase in the apparent rate of cross-bridge detachment. Received: 27 October 1995/Received after revision: 24 January 1996/Accepted: 30 April 1996     

Slip of rabbit striated muscle in rigor or AMPPNP

Summary Single glycerol-extracted rabbit psoas muscle fibres have been slowly extended either in rigor or in the unhydrolysable ATP analogue AMPPNP, and their sarcomere length, sarcomere structure and tension measured. The length of regularly arrayed sarcomeres, measured by optical diffraction, increased continuously as the muscle was stretched; the maximum sarcomere extension seen was approximately 6%. In the electron microscope sarcomeres from extended muscle fixed in rigor or AMPPNP remained regular in their internal structure, without rupture or obvious lengthening around the Z line. During steady extension at 0.024% per min the tension in the muscle fibre rose until it reached a limiting value [T _m ] when the sarcomeres had stretched by 0.8–1.6% and then remained constant with continued extension, while the sarcomeres continued to stretch. Provided that a novel form of preparation of the glycerol-extracted fibres was employed,T _m in rigor was a large fraction of the tension expected from an active isometric muscle fibre. In the presence of AMPPNPT _m was reduced by a factor of 2 to 3. Step extension by 0.08% at 5-min intervals gave the same pattern of mechanical response with similar values ofT _m . The isometric tension decay in the interval between the steps was very rapid at first and slowed continuously until the next step. The average speed of tension fall between 30 and 300 s after stretch was measured at each step and plotted relative to the tension in the muscle. The relationship approached linearity, although with a significant upward curvature at high tension. The proportionality constant of the rate of tension fall to tension was 4.5×10^–4 s^–1 in rigor and 9×10^–4 s^–1 in AMPPNP. These values are less than the apparent dissociation rate constants for acto-subfragment-1 or acto-heavy meromyosin under similar conditions (Marston, 1982). These results indicate that interfilament slip does occur in rabbit skeletal muscle both in rigor and in AMPPNP, but that it is much slower that that predicted from the behaviour of the isolated proteins, as if the myosin heads interacted so as to obstruct their detachment.     

Single turnover of cross-bridge ATPase in rat muscle fibers studied by photolysis of caged ATP

A mechanical study on skinned rat psoas muscle fibers was performed at about 16°C with X-ray diffraction and caged-ATP photolysis. The amount of photoreleased ATP was set <0.2 mM for analysis of a `single turnover' of the cross-bridge ATPase. With regard to the phase of activation, the results under the single turn-over condition were generally consistent with previous results obtained with larger amount of photoreleased ATP. Formation of the ADP-rigor state was mechanically monitored by the 90° out-of-phase component of stiffness at 500 Hz, which was elevated on activation and then decreased to zero with a half-time of 0.2–0.3 s. Intensity changes of the X-ray reflections (e.g. equatorial reflections, actin layer lines and a myosin meridional reflection) indicated that a large number of cross-bridges returned to the rigor structure with a half-time of 0.5–0.7 s. During this phase, tension did not increase but slowly decreased with a half-time of about 1.0 s. The in-phase stiffness increased only 20–30% at the most. These results indicate that, even if the number of cross-bridges formed at any moment during full contraction is small, they can interact with actin and form rigor bonds with a rate of 1 s^–1. The force developed in the rigor formation is probably lost due to the presence of rigor bridges and compliance in the preparation.     

Role of the N-terminal negative charges of actin in force generation and cross-bridge kinetics in reconstituted bovine cardiac muscle fibres

Mutant yeast actins were used to determine the role of actin's N-terminal negative charges in force generation. The thin filament was selectively removed from bovine cardiac skinned muscle fibres by gelsolin, and the actin filament was reconstituted from purified G-actin. In this reconstitution, yeast wild-type actin (2Ac: two N-terminal negative charges), yeast mutant actins (3Ac and 4Ac), and rabbit skeletal muscle actin (MAc) were used. The effects of phosphate, ATP and ADP on force development were studied at 25°C. With MAc, isometric tension was 77% of the initial tension owing to the lack of a regulatory system. With 2Ac, isometric tension was 10% of the initial tension; with 3Ac, isometric tension was 23%; and with 4Ac, isometric tension was 44%. Stiffness followed a similar pattern (2Ac < 3Ac < 4Ac < MAc). A similar trend was observed during rigor induction and relaxation. Sinusoidal analysis was performed to obtain the kinetic constants of the cross-bridge cycle. The results showed that the variability of the kinetic constants was ≤ 2.5-fold among the 2Ac, 4Ac and MAc muscle models. When the cross-bridge distribution was examined, there was no significant reapportionment among these three models examined. These results indicate that force supported by each cross-bridge is modified by the N-terminal negative charges of actin, presumably via the actomyosin interface. We conclude that two N-terminal negative charges are not adequate, three negative charges are intermediate, and four negative charges are necessary to generate force.     

The actin-binding cleft: functional characterisation of myosin II with a strut mutation

The myosin cross-bridge has two essential properties: to undergo the “power stroke” and to bind and release from actin – both under control of ATP binding and hydrolysis. In the absence of ATP the cross-bridge binds to actin with high affinity: the binding of ATP causes rapid release of the cross-bridge from actin. The actin binding-site is split by a deep cleft that closes on strong binding to actin. The cleft is straddled by a short polypeptide known as the “strut”. In the following we summarise the structural basis of the power stroke and the control of actin affinity and then present data on the effects on actin affinity of replacing the strut by a flexible linker.     

Mechanical and kinetic effects of shortened tropomyosin reconstituted into myofibrils

The effects of tropomyosin on muscle mechanics and kinetics were examined in skeletal myofibrils using a novel method to remove tropomyosin (Tm) and troponin (Tn) and then replace these proteins with altered versions. Extraction employed a low ionic strength rigor solution, followed by sequential reconstitution at physiological ionic strength with Tm then Tn. SDS-PAGE analysis was consistent with full reconstitution, and fluorescence imaging after reconstitution using Oregon-green-labeled Tm indicated the expected localization. Myofibrils remained mechanically viable: maximum isometric forces of myofibrils after sTm/sTn reconstitution (control) were comparable (~84%) to the forces generated by non-reconstituted preparations, and the reconstitution minimally affected the rate of isometric activation (k _act), calcium sensitivity (pCa₅₀), and cooperativity (n _H). Reconstitutions using various combinations of cardiac and skeletal Tm and Tn indicated that isoforms of both Tm and Tn influence calcium sensitivity of force development in opposite directions, but the isoforms do not otherwise alter cross-bridge kinetics. Myofibrils reconstituted with Δ23Tm, a deletion mutant lacking the second and third of Tm’s seven quasi-repeats, exhibited greatly depressed maximal force, moderately slower k _act rates and reduced n _H. Δ23Tm similarly decreased the cooperativity of calcium binding to the troponin regulatory sites of isolated thin filaments in solution. The mechanisms behind these effects of Δ23Tm also were investigated using P _i and ADP jumps. P _i and ADP kinetics were indistinguishable in Δ23Tm myofibrils compared to controls. The results suggest that the deleted region of tropomyosin is important for cooperative thin filament activation by calcium.     

Electrical and mechanical activities of frog heart during energetic deficiency

Summary Metabolic inhibition of frog heart by cyanide (3 × 10^–3 m) induced a drop in tension before any significant alteration occurred in the action potential or the slow inward current. The decline in electrical activity appeared later. After 20 min in cyanide solution at a time when both tension andI _slow were reduced, the addition of adrenaline (5 × 10^–7 m) greatly increased the slow inward current whereas tension only exhibited a partial and slow recovery. In both types of experiment the absence of correlation between the amount of inward Ca²⁺ current and the tension suggested the existence of a regulatory mechanism other than this current. It was not possible to correlate the fall in tension (which was parallel to the drop in creatine phosphate) with the reduction in ATP. ATP only decreased during application of both cyanide (CN^–) and iodoacetic acid (IAA). Quick-stretch and quick-release experiments, conducted during the tension which developed in the presence of CN^– and IAA, showed that the mechanical activity did not result from Ca²⁺-activated tension but from rigor tension, implying a marked deficiency in local ATP. A shortage of the energy available for myofibrillar ATPase activity might be one explanation for the drop in peak tension during metabolic inhibition and the rise in resting tension observed after more severe inhibition might be a shortage of the energy-rich phosphates available for myofibrillar ATPase activity.     

Characterizations of myosin essential light chain’s N-terminal truncation mutant Δ43 in transgenic mouse papillary muscles by using tension transients in response to sinusoidal length alterations

Cross-bridge kinetics were studied at 20 °C in cardiac muscle strips from transgenic (Tg) mice expressing N-terminal 43 amino acid truncation mutation (Δ43) of myosin essential light chain (ELC), and the results were compared to those from Tg-wild type (WT) mice. Sinusoidal length changes were applied to activated skinned papillary muscle strips to induce tension transients, from which two exponential processes were deduced to characterize the cross-bridge kinetics. Their two rate constants were studied as functions of ATP, phosphate (Pi), ADP, and Ca²⁺ concentrations to characterize elementary steps of the cross-bridge cycle consisting of six states. Our results demonstrate for the first time that the cross-bridge kinetics of Δ43 are accelerated owing to an acceleration of the rate constant k ₂ of the cross-bridge detachment step, and that the number of strongly attached cross-bridges are decreased because of a reduction of the equilibrium constant K ₄ of the force generation step. The isometric tension and stiffness of Δ43 are diminished compared to WT, but the force per cross-bridge is not changed. Stiffness measurement during rigor induction demonstrates a reduction in the stiffness in Δ43, indicating that the N-terminal extension of ELC forms an extra linkage between the myosin cross-bridge and actin. The tension-pCa study demonstrates that there is no Ca²⁺ sensitivity change with Δ43, but the cooperativity is diminished. These results demonstrate the importance of the N-terminal extension of ELC in maintaining the myosin motor function during force generation and optimal cardiac performance.     

High frequency characteristics of elasticity of skeletal muscle fibres kept in relaxed and rigor state

Summary The viscoelastic properties of crossbridges in rigor state are studied by means of application of small length changes, completed within 30 s, to isometric skinned fibre segments of the iliofibularis muscle of the frog in relaxed and rigor state and measurement of the tension response. Results are expressed as a complex Young's modulus, the real part of which denotes normalized stiffness, while the imaginary part denotes normalized viscous mechanical impedance. Young's modulus was examined over a wide frequency range varying from 5 Hz up to 50 kHz. Young's modulus can be interpreted in terms of stiffness and viscous friction of the half-sarcomere or in terms of elastic changes in tension and recovery upon a step length change.The viscoelastic properties of half-sarcomeres of muscle fibre segments in rigor state showed strong resemblance to those of activated fibres in that shortening a muscle fibre in rigor state resulted in an immediate drop in tension, after which half of the drop in tension was recovered. The following slower phases of tension recovery—a subsequent drop in tension and slow completion of tension recovery—as seen in the activated state, do not occur in rigor state. The magnitude of Young's moduli of fibres in rigor state generally decreased from a value of 3.12×10⁷ N m^-2 at 40 kHz to 1.61×10⁷ N m^-2 at about 100 Hz.Effects of increased viscosity of the incubation medium, decreased interfilament distance in the relaxed state and variation of rigor tension upon frequency dependence of complex Young's modulus have been investigated. Variation of tension of crossbridges in rigor state influenced to some extent the frequency dependence of the Young's modulus. Recovery in relaxed state is not dependent on the viscosity of the medium. Recovery in rigor is slowed down at raised viscosity of the incubation medium, but less than half the amount expected if viscosity of the medium would be the cause of internal friction of the half-sarcomere. Internal friction of the half-sarcomere in the relaxed fibre at the same interfilament distance as in rigor is different from internal friction in rigor. It will be concluded that time necessary for recovery in rigor cannot be explained by friction due to the incubation medium. Instead, recovery in rigor expressed by the frequency dependence of the Young's modulus has to be due to intrinsic properties of crossbridges. These intrinsic properties can be explained by the occurrence of state transitions of crossbridges in rigor. Similarity of Young's modulus of fibre segments in the activated state and in rigor in the frequency range above 5 kHz strongly suggests that the same state transitions occur in force generating crossbridges in the activated fibre.     

Comparison of cross-bridge cycling kinetics in neonatal vs. adult rat ventricular muscle

The developmental shift in contractile protein isoform expression in the rodent heart likely affects actin-myosin cross-bridge interactions. We compared the Ca²⁺ sensitivity for force generation and cross-bridge cycling kinetics in neonatal (postnatal days 0–3) and adult (day 84) rats. The force-pCa relationship was determined in Triton-X skinned muscle bundles activated at pCa 9.0 to 4.0. In strips maximally activated at pCa 4.0, the following parameters of cross-bridge cycling were measured: (1) rate of force redevelopment following rapid shortening and restretching (k_tr); and (2) isometric stiffness at maximal activation and in rigor. The fraction of attached cross-bridges (_fs) and apparent rate constants for cross-bridge attachment (f_app) and detachment (g_app) were derived assuming a two-state model for cross-bridge cycling. Compared to the adult, the force-pCa curve for neonatal cardiac muscle was significantly shifted to the left. Neonatal cardiac muscle also displayed significantly smaller _fs, slower k_tr and f_app; however, g_app was not significantly different between age groups. These data indicate that weaker force production in neonatal cardiac muscle involves, at least in part, less efficient cross-bridge cycling kinetics.