Insect Fibrillar Muscle Research Articles

Role of cross-bridge distortion in the small-signal mechanical dynamics of insect and rabbit striated muscle.

The mechanism of the active tension response of insect fibrillar muscle to step changes and small oscillations of length was re-investigated, following White's demonstration (1983) that the high relaxed stiffness evidently persists during activation and cannot be neglected as had previously been assumed. White's result makes earlier explanations of the small-signal response untenable; the experimental and theoretical studies described here lead to a new class of explanations at the cross-bridge level. The response of an activated muscle to a fast stretch consists of a synchronous tension increase that is followed first by a rapid decay of tension and then by a delayed rise ('stretch activation'). It was shown in glycerinated fibre preparations from the water bug and the bumblebee that subtraction of the relaxed tension response from the active response results in a prominent undershoot of the tension level preceding the step, before the delayed rise of tension. The responses of the same fibres to sinusoidal oscillations, in the frequency range 1-150 Hz, showed an equivalent behaviour, with the active locus circling the relaxed locus in a Nyquist plot, as described by Machin & Pringle (1960). Stiffness was determined during the tension response to a large step (of 1%) by recording the immediate change of tension to a small test step (0.2%), applied at various times after the conditioning step. In the majority of preparations stiffness remained constant or increased during the undershoot of tension. Step and sinusoidal responses with the above features cannot be explained at all by an active component resembling a simple exponential delay. We show, however, that such features are predicted if certain small-signal effects of cross-bridge distortion (previously and erroneously assumed negligible in insect flight muscle for the small-signal case) are incorporated in models of the cross-bridge cycle. Two alternative hypotheses for the effects of distortion are examined: (i) distortion-induced detachments and (ii) distortion-modulated transitions among multiple attached states (Huxley & Simmons, 1971). For the first we also show that the results do not differ qualitatively whether one assumes strain, interfilament displacement or 'bridge recruitment' as the physical correlate of stretch activation. Both of the above explanations account, at least qualitatively, for the observed rapid decay and undershoot of tension following a step increase of length, and for the circling of the active Nyquist-plot loci about the passive locus. The explanation based on distortion-induced detachments, however, appears to be inconsistent with the stiffness measurements.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a connecting filament protein in insect fibrillar flight muscle

A major component on sodium dodecyl sulfate-containing gels of solubilized isolated Z-discs, purified from honeybee flight muscle, migrates with an apparent molecular weight of 360,000. Antibodies to this high molecular weight polypeptide have been prepared by injecting rabbits with homogenized gel slices containing the protein band. With indirect immunofluorescence microscopy these antibodies are localized to a region extending from the edge of the Z-band to the A-band in shortened or stretched sarcomeres. Similarly, glycerinated flight muscle treated with antiserum and prepared for electron microscopy shows enhanced density from the ends of the thick filaments to the I-Z junction regardless of sarcomere length. Evidence indicates that antiserum is directed toward a structural protein of connecting filaments, which link thick filaments to the Z-band in insect fibrillar muscle, rather than to a thin filament component. In Ouchterlony double-diffusion experiments a single precipitin band is formed when antiserum is diffused against solubilized Z-discs; no reaction occurs between antiserum and proteins from native thin filaments prepared from honeybee flight muscle. Further, antibody stains the I-band in flight muscle fibrils from which thin filaments are removed. Finally, honeybee leg muscle myofibrils, in which connecting filaments have not been observed, are not labelled with antibody. Since antibody binds to the short projections which extend from the flat surfaces of isolated Z-discs, these projections are assumed to be remnants of connecting filaments and the source of the 360,000 M r protein. The amino acid composition of this high molecular weight material, purified by Sepharose chromatography, is presented. The protein has been named “projectin”.

Activation of the contractile system of insect fibrillar muscle at very low concentrations of Mg2+ and Ca2+.

The mechanical properties of single fibres and fibre bundles of glycerinated dorsal longitudinal muscle from lethocerus maximus were investigated in ATP-salt solutions containing only trace concentrations of free Ca2+ (pCa>9). A reduction in the magnesium concentration (pMg ∼ 7) resulted in an increase in the instantaneous stiffness of glycerinated insect flight muscle fibres, though very little accompanying tension was developed. Stiffness was measured either using small amplitude sinusoidal length changes of high frequency (1 kHz) or rapid rectangular form length changes. The ratio of stiffness to tension in solutions free of added magnesium and calcium was equal to or greater than that obtained from the tissue in the rigor state, and much larger than that obtained in the presence of both magnesium and calcium. Extrapolation of the linear part of the change-tension relationship (obtained during rapid length changes completed within 0.3 ms) back to zero tension indicated that the elastic elements of attached crossbridges were less extended under conditions of Mg2+-deprivation than during Ca-activation in Mg2+-rich solution. Following a quick stretch a delayed tension development similar to that obtained in the presence of magnesium and calcium ions was observed. The rise in tension was delayed with respect to the accompanying rise in stiffness and reached a peak value after about 2 s. Similar tension transients followed a subsequent release. The possibility that an unusually slow corss-bridge cycle might be responsible for these slow transients was suggested by the finding that the fibres showed a very low ATPase activity under these conditions which could be slightly activated by stretches. On increasing the free Ca2+ concentration during magnesium deprivation, the time course of the stretch induced tension transients became faster, while stiffness and the steady state tension rose to reach a ‘high tension state’ at aboutpCa 6.5.

Stiffness and tension during and after sudden length changes of glycerinated single insect fibrillar muscle fibres.

Rapid length changes were applied (within 0.2 ms or 0.4 ms) to single isometrically contracted glycerol extracted muscle fibres of the dorsal longitudinal muscle ofLethocerus maximus suspended in an Ca2+ and ATP containing solution at 20–23‡ C. Force transients and the fibre stiffness were measured during and after rapid length changes.

The Croonian Lecture, 1977. Stretch activation of muscle: function and mechanism.

Current theories about the mechanism of muscular contraction suppose that the level of enzymic and contractile activity is controlled by the intracellular concentration of calcium ions, the degree of overlap between the myosin and actin filaments and the rate of relative sliding of the filaments. It is now known that in most or all muscles there is a further direct influence of mechanical conditions, usually called stretch activation; changes of length lead to a delayed change of active tension. The effect is large and functionally significant in insect fibrillar flight muscle and in mammalian heart muscle; it is present, but small, in vertebrate skeletal muscle, which probably accounts for its late discovery. In insect fibrillar flight muscle, the delayed tension is responsible for the rhythmic mechanical activity during flight. In mammalian heart muscle it may play a rôle in Starling’s Law. In insect fibrillar muscle, extension produces a maintained increase in actomyosin ATPase and active tension; in vertebrate skeletal muscle, stretch activation is a transient phenomenon. Mammalian heart muscle shows greater maintenance of stretch activation than skeletal muscle; the duration of higher ATPase activity has not yet been determined. The effective mechanical parameter is not overall strain but is probably the strain on an internal structure related to overall stress. Various lines of evidence point to the myosin filament as the location of the sensor. A considerable degree of molecular synchronization occurs during natural insect flight.

Temperature and amplitude dependence of tension transients in glycerinated skeletal and insect fibrillar muscle.

1. Quick stretches and releases were applied to small bundles of glycerinated fibres of rabbit psoas and insect fibrillar flight muscle. The resulting tension changes were recorded at various temperatures and amplitudes of length change. The results from the two preparations had many features in common. At temperatures near 0 degrees C the asymmetry of the initial tension recovery after stretch and release originally reported in living frog fibres by Huxley & Simmons (1971 alpha) was very obvious. 2. The complete tension course could be described as an elastic change occurring simultaneously with the length change followed by recovery consisting of the sum of a number of exponential terms. These terms usually corresponded to the phases discernible without curve fitting, but in some cases a monotonic rise or fall of tension was seen to consist of two components only after curve fitting. 3. After either stretch or release there was a phase of rapid tension recovery towards the value before the length change. The rate constant of this phase increased as the amplitude of stretch or release was increased to about 2 nm/half sarcomere. At higher amplitudes it remained nearly constant 4. At temperatures near 0 degrees C there was a second and much slower continuation of the recovery after stretch. The rate constant of this second phase was much more sensitive to temperature than that of the first phase and it became slower with increasing amplitude of stretch. As the temperature was raised the speed of the second phase approached the speed of the first phase so that at room temperatures the initial tension recovery after stretch and release was nearly symmetrical. 5. Under many conditions these processes were followed by a change in the opposite direction, the 'delayed tension' described by earlier workers. This third phase of tension change had about the same temperature sensitivity as the second phase of the recovery seen after stretch. The tension due to stretch activation was not maintained in rabbit muscle, resulting in a fourth possible phase, a recovery of tension towards the value before the length change. This was absent or of low amplitude in insect flight muscle. 6. We interpret these tension changes on the basis of an extension of the non-linear model described by White & Thorson (1972). The elastic tension change and the initial fast recovery are both supposed to be properties of the attached cross-bridges, whilst the slower recovery is considered to be due to the detachment of cross-bridges which happened to be attached at the instant the length change was applied. The delayed tension reflects the approach to equilibrium of the number of attached bridges, changed by an effect of muscle length on the attachment rate. The fact that the delayed tension is not maintained in rabbit psoas muscle may be due to the effect of length on attachment rate being transitory.

Is the chemomechanical energy transformation reversible?

In glycerol-extracted insect fibrillar muscle suspended in ATP salt solution the incorporation of 32Pi into ATP was studied during the performance of positive or negative oscillatory work and under a variety of mechanical and ionic conditions. An increase in calcium ion concentration from 10(-8)--10(-5) M increased the incorporation rate in proportion to the increase in ATPase activity, mean tension and immediate stiffness, which is a measure of the extent of actin-myosin interaction. Sinusoidal stretches (at 1% Lo) performed at 5 Hz induced the fibres to perform optimal positive oscillatory work and it caused a doubling of the incorporation rate (ant ATPase activity). A decrease or increase of the frequency below or above the optimum of 5 Hz always decreased the power output as well as the incorporation rate which, however, was still noticeable even under conditions where work was done on the fibres. A similar frequency dependence was found when square-wave rather than sinusoidal stretches were applied and this effect could be related to the finding that the rate of stretch-induced incorporation was highest shortly after stretching and then declined to low values (after about 100 ms). These results suggest the formation of an energy-rich intermediate (actomyosin-ADP?) during the contraction process induced by stretching and this intermediate must be assumed to accumulate transiently after stretching.

Calcium and tension-dependent changes in the actin filament structure of insect fibrillar muscle

Calcium and tension-dependent changes in the actin filament structure of insect fibrillar muscle

The velocity-dependence of myosin cross-bridge movement and tension development in oscillatory contractions of insect fibrillar muscle

Rontgenstrukturanalysen zeigen, dass die Beziehung zwischen sinusoidaler Kraftententwicklung und der Kinetik der Myosinbruckenzyklen von der Oszillationsgeschwindigheit abhangt.

Changes in myosin cross-bridge attachment during oscillatory contractions of insect fibrillar muscle

FEBS LettersVolume 40, Issue 1 p. 45-48 Full-length articleFree Access Changes in myosin cross-bridge attachment during oscillatory contractions of insect fibrillar muscle R.A. Chaplain, R.A. Chaplain Department of Biocybernetics, Advanced College of Technology, Magdeburg, Kleiberweg 14, GDRSearch for more papers by this authorB. Honka, B. Honka Department of Biocybernetics, Advanced College of Technology, Magdeburg, Kleiberweg 14, GDRSearch for more papers by this author R.A. Chaplain, R.A. Chaplain Department of Biocybernetics, Advanced College of Technology, Magdeburg, Kleiberweg 14, GDRSearch for more papers by this authorB. Honka, B. Honka Department of Biocybernetics, Advanced College of Technology, Magdeburg, Kleiberweg 14, GDRSearch for more papers by this author First published: March 15, 1974 https://doi.org/10.1016/0014-5793(74)80890-2Citations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume40, Issue1March 15, 1974Pages 45-48 ReferencesRelatedInformation

Mechanical and electrical oscillations in cardiac muscle of the turtle.

During contractures of the turtle ventricle rapid changes in length induce sinusoidal oscillations under isotonic conditions. They are due to delayed responses to stretching and release, which can be demonstrated also under isometric conditions. Oscillations of two distinct frequencies are produced under different conditions and are distinguished as high- and low-frequency oscillations. In depolarized muscles the frequency is such that the duration of one cycle is about the same as that of a normal twitch, while in high-Ca solutions the duration can be the same as in high-K solutions or about six times lower. As reported previously, twitches are followed by weak mechanical and electrical oscillations. Their frequency agrees with the high-frequency oscillations. The same effects can also be induced by stretching and release. It is suggested that the phenomena observed are due to feedback mechanisms which originate in the contractile mechanism. The high-frequency oscillations are similar to those observed previously in other muscles, particularly insect fibrillar muscle, and are not due to changes in Ca concentration. The other mechanisms involve the membrane and possibly the intracellular Ca stores.

The effects of fibre length and calcium ion concentration on the dynamic response of glycerol extracted insect fibrillar muscle.

1. The property of insect fibrillar muscle which enables it to oscillate continuously when it is connected to a resonant load is the delayed activation by stretch of contractile activity. The dynamic response has been measured at different fibre lengths and at different calcium ion concentrations, to see what effects these conditions have on the magnitude and rate constant of the delayed tension.2. Bundles of ten fibres of the dorsal longitudinal muscle of the water bug, Lethocerus cordofanus, which had been in glycerol for less than 5 days were used. Graded activation was obtained with buffered calcium ion concentrations between 10(-7) and 10(-5)M.3. A computer-controlled apparatus was used to measure the dynamic mechanical properties of the muscle fibres. This allowed many measurements to be made on the same preparation. Further computer programs analysed the Nyquist plots produced by the experiment.4. When the mean length of a fibre bundle was increased, the magnitude of the delayed tension was increased (for a constant amplitude of sinusoidal length change), the rate constant was unaltered, and the stiffness of the fibres was increased. When the calcium ion concentration was raised, the magnitude of the delayed tension was increased, the rate constant increased, and the stiffness of the fibres fell. Calcium activation and stretch activation are thus clearly separable in their effects, and so the mechanisms must be separate.5. The various different effects of calcium cannot be explained by any simple model of activation, for example, an on-off switch mechanism controlling the number of bridges in action.6. The stretch-induced activity is proportional to a power of the length of two or greater and this non-linearity aids the efficient operation of the oscillatory mechanism.

Persistence of adenylate kinase and other enzymes in glycerol extracted muscle.

The use of glycerol extracted muscle allows mechanical studies on the contractile mechanism to be carried out under defined biochemical conditions. It is important to know whether enzymes, other than the myofibrillar ATPase, which act on adenine nucleotides are present in this preparation. Therefore, the effects of glycerol extraction were studied on adenylate kinase and arginine kinase activities in insect fibrillar muscle and on adenylate kinase, creatine kinase and AMP deaminase activities in rabbit psoas muscle. All these activities decreased during glycerol extraction. A substantial part of the activity was lost in the first day of glycerol extraction, after which a very slow rate of loss occurred. There was some indication of a preferential loss from the soluble fraction of the fibres but this was insufficient to explain the biphasic loss. Measurements of the total activity of adenylate kinase in the fibres plus the glycerol medium suggested that some inactivation had occurred. It is also possible that the outer fibres in a bundle are extracted more rapidly than are the inner fibres. All three effects are probably playing a part but we are unable to offer a complete quantitative explanation of the results.

Feedback in the contractile mechanism of the frog heart.

Shortening causes a transient decrease, extension an increase, in activity during contractures of the frog ventricle induced by high Ca or by isosmotic K solution. This is shown by the fact that, after the immediate passive shortening, the muscle is extended under isotonic conditions when the load is diminished, and that under isometric conditions quick release causes first a rapid drop, then a further, much slower, fall of tension. Increasing the load or stretching induce the opposite effects. At low temperatures all rapid changes in length produce oscillations of low frequency. These responses are due to a sensitive feedback mechanism similar to that previously demonstrated for insect fibrillar muscle. That this mechanism comes into play in the heart under normal conditions and controls the time-course of the twitch is demonstrated by the observation that relaxation begins earlier the greater the shortening. Thus, during afterloaded isotonic twitches the onset of relaxation is advanced as the load is diminished.

Mechanical activation and isometric oscillation in insect fibrillar muscle.

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 (Q10) 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 A, 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.

Effects of inorganic phosphate on the contractile mechanism.

Inorganic phosphate in millimolar concentrations reduces contractile tension, but increases the speed and the oscillation frequency of glycerinated insect fibrillar muscle, while power output and ATPase are little affected.

Activation by ADP and the correlation between tension and ATPase activity in insect fibrillar muscle.

The effect of ADP on the tension and ATPase activity of glycerinated fibres of the flight muscles of the giant water bugLethocerus cordofanus has been investigated. Both increased, by a variable amount, when the ADP concentration was raised to 1 mM, but the increase was reversed when the concentration was raised further. In another series of experiments, the effect of temperature on the ATPase activity and tension was measured. There was a positive correlation between ATPase activity and tension in both types of experiment, the slopes being similar to those reported by other workers when calcium ion concentration or mean muscle length were altered. The positive feedback resulting from the increase of ATPase activity caused by ADP explains at least in part the variability of the results, and provides a possible explanation of the “high tension state” in glycerinated fibrillar muscle.

Mechanical properties of insect fibrillar muscle at large amplitudes of oscillation

Glycerol-extracted fibres from Lethocerus flight muscle were subjected to large-amplitude sinusoidal oscillation in a solution containing Mg-ATP and Ca 2+ at room temperature, under conditions designed to eliminate the effects of diffusion of nucleotides into and out of the fibre. They produced work up to 4 to 8 % peak-to-peak oscillation amplitude at 1.5 to 2.5 Hz and up to lower amplitudes at higher frequencies; freshly extracted fibres produced work at higher amplitudes than aged fibres. The mean tension in the muscle was shown to be critical for work production. Provided this parameter was maintained constant, the mean fibre length, Ca 2+ concentration and ADP concentration could be varied considerably without loss of oscillatory work; these factors are therefore not in themselves critical for oscillation. The tension/length relationship of relaxed fibres is non-linear at high amplitudes of oscillation. Oscillatory tension/length loops are not elliptical at high amplitude and change shape when mean tension and frequency are changed. Linear control theory cannot be used to analyse performance at high amplitudes. At the upper limit of mechanical performance the oscillation amplitude and work-per-cycle performed by the freshly extracted fibres were similar to those of live flight muscle, and the active tension generated was similar to that from other maximally activated striated muscles. Calculation suggests that most or all of the myosin in the muscle was activated in each cycle of oscillation. The limits of performance are therefore probably present in life and are characteristics of the entire contractile material; their possible significance is discussed.

Tension transients in glycerol-extracted fibres of insect fibrillar muscle (Lethocerus maximus).

Mit Wasser-Glyzerin extrahierte fibrillare Muskelfasern (Fasermodelle) vonLethocerus maximus zeigen im ATP-Bad nach plotzlicher Dehnung das Phanomen der Dehnungsaktivierung, gefolgt von einer gedampften, temperaturabhangigen isometrischen Oszillation der Kontraktionsspannung. Die Frequenz entspricht offenbar der Eigenfrequenz zyklisch tatiger « KontraktionsbrUcken » zwischen Aktin und Myosin.

Activation in a Skeletal Muscle Contraction Model with a Modification for Insect Fibrillar Muscle

A sliding filament model for muscle contraction is extended by including an activation mechanism based on the hypothesis that the binding of calcium by a regulating protein in the myofibrils must occur before the rate constant governing the making of interactions between cross-bridges and thin filament sites can take on nonzero values. The magnitude of the rate constant is proportional to the amount of bound calcium. The model's isometric twitch and rise of force in an isometric tetanus are similar to the curves produced by real muscles. It redevelops force after a quick release in an isometric tetanus faster than the initial rise. Quick release experiments on the model during an isometric twitch show that the "active state" curve produced is different from the postulated calcium binding curve. The force developed by the model can be increased by a small quick stretch delivered soon after activation to values near the maximum generated in an isometric tetanus. Following the quick stretch, the force remains near the tetanic maximum for a long time even though the calcium binding curve rises to a peak and subsequently decays by about 50%. The model satisfies the constraint of shortening with a constant velocity under a constant load. Modifications can be made in the model so that it produces the delayed force changes following step length changes characteristic of insect fibrillar muscle.