Insect Fibrillar Muscle Research Articles

Changes of adenosine triphosphatase activity and tension with fibre elongation in glycerinated insect fibrillar flight muscle

1. The relationship between fibre elongation and the CA2+-activated ATPase of glycerinated insect fibrillar muscle has been studied. 2. The A-filaments in insect fibrillar muscle are directly strained in a Ca2−-free solution in the absence of permanent actin-myosin interactions. Strain increases the sensitivity of the fibres to low levels of Ca2+. 3. Both ATPase and tension increase up to a fibre extension of 10%; above this fibre length no further strain can be applied to the A-filaments. Between 10% and 20% stretch both tension and ATPase reach a plateau. 4. At fibre elongations greater than 20% both fibre tension and ATPase activity decrease again corresponding to the reduction in the overlap between the A-and I-filaments.

Energetics and "efficiency" in the isolated contractile machinery of an insect fibrillar muscle at various frequencies of oscillation.

Glycerol extracted fibres from the dorsal longitudinal muscle ofLethocerus maximus represent the “isolated” contractile machinery and they produce oscillatory power in ATP salt solution when they are driven to oscillate at frequencies greater than 0.5 c/sec by sinusoidal stretch and release. Increasing the frequency increases the ATPase activity (oscillation induced or “dynamic” extra ATPase) and the output of power. But in previous experiments, an increase in frequency exceeding about 4 c/sec often leads to a complete loss of power output and to the development of the “high-tension state”, especially when the free calcium concentration was high (10−5 M), at high temperatures (above 25°C) or at large amplitudes of oscillation. Avoiding the “high-tension state” by supplying the glycerinated fibres with up to 15 mM Mg-ATP or 0.1 mg/ml myokinase we are now able to show that the rate of ATP splitting and the rate of doing work are both optimal when the frequency was 20–25 c/sec (the wingbeat frequency of living Lethocerus) at 10−5M Ca++ and 30°C, even at physiological oscillation amplitude (about 2–3% of the fibre length). The power output is then of the same order of magnitude as in live insect flight muscle. Above the optimum frequency, both power output and ATPase activity decrease in a parallel fashion and they vanish completely at about 60 c/sec. Decreasing either the free calcium concentration or the temperature reduces the optimum frequency. The parallelism of the frequency dependence of the ATPase activity and power output under various conditions is a consequence of the rather constant ratio of the rate of doing work and the rate of ATP splitting, both of which depend on the rate of actin-myosin interaction. At high calcium concentration the ratio or the mechanochemical coefficient is about 3–4 kcal/mole of ATP split (i.e. 50%), at 10 c/sec oscillation but the efficiency is much less at 15 or 2 c/sec, indicating a less tight chemo-mechanical coupling.

Activation of the myofibrillar ATPase activity by extension of glycerolextracted insect fibrillar muscle.

The myofibrillar ATPase activity of glycerol extracted insect fibrillar muscle fibres (Dorsolongitudinal muscle, Lethocrrus) suspended in ATP-salt solution was determined in dependence of fibre length. Little change occurred between 70 and 100%L 0. But stretching by only 2–3%L 0 nearly doubled the ATPase activity and the contractile tension. The proportional increase in tension an ATPase activity in the range of 100–103%L 0 (30 pmoles ATP split for 1 dyne by 1 cm fibre during 1 min) observed after stretching suggests an increase in the extent of actinmyosin interaction, inspite of the fact that the actin-myosin overlap zone then slightly decreases. The relation between tension increase and ATPase activity increase is nearly the same after stretching or after raising the free calcium concentration; nonetheless, the stretch induced activation is apparently not due to an increase in the calcium affinity of actomyosin (as suggested byChaplain) because stretch activation is observed even at ‘enzyme saturating’ concentrations of 10−5 M Ca2+. Furthermore the sigmoidal relationship between ATPase activity (or tension) and p Ca and in particular the calcium concentration required for 50% activation is similar in stretched and unstretched fibres, in myofibrils and in actomyosin. The fibre preparation may be stretched in ‘relaxing solution’ containing less than 10−8 M Ca++ (i.e. in the absence of actin-myosin crosslinking), but the ATPase activity increase following stretching is then only observed after increasing the [Ca++] to ≳ 10−7 M; it is correlated with passive tension in relaxing solution rather than with the extent of stretch, suggesting that the element responding to a length change is apparently the passive tension bearing myosin filament which is attached to the Z-line in insect flight muscle.

The coupling of poweroutput and myofibrillar ATPase activity in glycerol-extracted insect fibrillar muscle at varying amplitude of ATP-driven oscillation.

Glycerol extracted fibre bundles or single fibres from the dorsolongitudinal muscle of Lethocerus maximus were suspended in ATP-saltsolution; they were then held isometrically or they were sinusoidally stretched and released about a mean length (102%L0) at an oscillation amplitude of 1–4% of the length. At very low frequencies (less than 0,2 cycles/sec) the ATPase activity remained the same as under static conditions at the mean length since the activity increase produced by stretch was compensated by the activity reduction during release. At frequencies approaching 2–3 cycles/sec the ATPase activity greatly exceeded the activity level under static conditions (at the mean fibre length and even at the extended length) by an amount (Extra-ATPase activity) depending on the oscillation amplitude. At this frequency sinusoidal tension changes lagged behind sinusoidal length changes, indicating that the fibre preparation was producing oscillatory power (about 0.3 μ cal/min cm fibre). The power output was often proportional to the square of the oscillation amplitude and mostly proportional to the oscillation induced extra-ATPase activity, suggesting a close mechanochemical coupling in the sense of a biochemical ‘Fenn effect’. At an oscillation amplitude the of 3–4%L0 maximal rates of extra ATP-splitting were observed, which amounted to 1–3 molecules of ATP split per crossbridge during each oscillation cycle. The mechanochemical coefficient approached 2–3 kcal/mole ATP split, indicating and ‘efficiency’ of up to 30%. We suggest that power producing oscillation increases the rate of actin-myosin interactions and we conclude that — like living muscle — the isolated contractile machinery transforms chemical energy (from ATP) into mechanical work with a high efficiency, and that the amount of work done controls the extent of the chemical reaction which takes place.

Evidence from insect fibrillar muscle about the elementary contractile process.

Bundles of myofibrils prepared from the dorsal longitudinal flight muscles of giant water bugs show oscillatory contractile activity in solutions of low ionic strength containing ATP and 10(-8)-10(-7)M Ca(2+). This is due to delay between changes of length and changes of tension under activating conditions. The peculiarities of insect fibrillar muscle which give rise to this behavior are (1) the high elasticity of relaxed myofibrils, (2) a smaller degree of Ca(2+) activation of ATPase activity in unstretched myofibrils and extracted actomyosin, and (3) a direct effect of stretch on ATPase activity. It is shown that the cross-bridges of striated muscle are probably formed from the heads of three myosin molecules and that in insect fibrillar muscle the cycles of mechanochemical energy conversion in the cross-bridges can be synchronized by imposed changes of length. This material is more suitable than vertebrate striated muscle for a study of the nature of the elementary contractile process.

The effect of Ca 2+ and fibre elongation on the activation of the contractile mechanism of insect fibrillar flight muscle

1. The relationship between fibre length and the Ca 2+-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) of insect fibrillar muscle has been studied. 2. Low levels of Ca 2+ and small degrees of fibre extension have been found to increase the ATPase activity. Either factor influences the sensitivity of the material to the other factor. 3. In insect fibrillar muscle, where the A filaments are continuous with the Z line, it is possible to strain the A filaments directly in the absence of permanent actin-myosin interactions. 4. The higher ATPase activity as a result of fibre elongation is accompanied by an increase in Ca 2+ binding. 5. Small maintained length changes do not affect the rate of ATP breakdown catalyzed by glycerinated fibres from frog sartorius or rabbit psoas muscle. 6. The relevance of the findings to the mechanism of oscillatory contraction of insect fibrillar muscle is discussed.

Preparation and properties of the contractile element of insect fibrillar muscle.

Abstract Non-myofibrillar enzyme activities persist after glycennation of insect flight muscle. Preservation of mitochondrial structure and function in glycerol-extracted preparations complicates the study of direct effects on the myofibrillar contractile machinery. Non-myofibrillar adenosine triphosphatase and a significant amount of respiration and oxidatdve phosphorylation in the presence of added substrate persist in glycerol-extracted fibres of insect fibrillar muscle stored for 6 months at − 18 °C. The standard procedure used to extract actomyosin from myofibrils also extracts from the mitochondrial fraction a protein which shows the phenomenon of super-precipitation and undergoes viscosity changes in the presence of adenosine triphosphate and metal ions. Preparation of the true contractile structure of insect flight muscle therefore re-quires the removal of all non-myofibrillar activities. Reduction in the total number of mitochondria and of non-myofibrillar enzymic activity is achieved by treatment with the detergent Tween 80 subsequent to glycennation without impairing the mechanical performance of the fibres. This provides better experimental material for studies of the contractile system than the preparations commonly used. The fact that fibres can still perform oscillatory work after treatment with detergent and oligomycin confirms that the control mechanism for oscillation resides within the myofibrils.

Oscillatory contraction of insect fibrillar muscle after glycerol extraction

Insect fibrillar muscle can sustain oscillations in a mechanically resonant load because length changes of the active muscle are followed by delayed changes of tension. It has not been known whether this is an intrinsic property of the contractile mechanism, or whether it depends on the excitation-contraction coupling system. The object of the present investigations has been to decide between these two possibilities by examining the behaviour of glycerol-extracted fibrillar muscle from giant water bugs (HydrocyriusandLethocerus, Order: Hemiptera). The purpose of the glycerol extraction was to isolate the contractile protein system by selective destruction of cellular components other than the myofibrils. Preparations consisting of single muscle fibres or small bundles of fibres were immersed in buffered salt solutions containing adenosine triphosphate (ATP), magnesium and calcium. Activation of the fibres could be produced, and the level of activity controlled, by varying the Ca2+concentration, which was stabilized by the presence of a calcium buffer (ethylene glycol bis-(β-aminoethyl ether) -N, N' -tetraacetate). Activity was detected by tension devel­opment and by the fact that length changes were followed by delayed changes of tension: this was revealed by transient analysis, which showed that a sudden release or stretch was followed by a delayed change of tension; by sinusoidal analysis, which showed a phase lag of tension on length (driven oscillation experiments); and by the fact that the preparation could sustain oscillations in a resonant lever system by overcoming its external damping (free oscillation experiments). Attempts to raise the level of activity or the power output of the preparation sometimes produced a change in the mechanical properties of the muscle, characterized by a rise of tension and an alteration in the oscillatory behaviour (the ‘high tension’ state). Inadequate diffusion ofATPinto, or adenosine diphosphate (ADP) out of, the preparation was probably the cause of this phenomenon. Under the conditions of the present experiments, the threshold Ca2+concentration varied from 2 x 10-8m to 12 x 10-8m. However, the sensitivity of the preparation to Ca2+depended on the concentration of Mg2+, which had a weak antagonistic effect on the action of Ca2+, and on the mechanical conditions. The effect of muscle length was of particular importance, as stretching the fibre seemed to be equivalent to raising the Ca2+concentration. It is suggested that this effect might provide the control mechanism by which changes of muscle length lead to delayed changes of tension. From studies of the effect of varying the chemical conditions it is concluded that fluctua­tions in the levels of Ca2+, H+,ATP,ADP, and inorganic phosphate are not responsible for the oscillatory behaviour of the glycerinated preparation. The mechanical properties of the living and glycerinated muscle are compared; it is concluded that raising the Ca2+concentra­tion is equivalent to stimulation of the living muscle, and that the oscillatory contractions produced in the two cases are essentially the same. The glycerol-extraction procedure was not entirely successful in producing structural isolation of the contractile protein system, but possible participation of other cellular components in the oscillatory contraction is con­sidered to have been excluded by the chemical conditions (in particular, by the presence of the calcium buffer). It is therefore concluded that the delay between change of length and change of tension is an intrinsic property of the contractile protein system.

Muscular Activity During Preparation For Flight in A Beetle

ABSTRACT The sound produced during the pre-flight activity of the water beetle Acilius sulcatus (L.) consists of a train of pulses, implying that there is a click mechanism present in the thorax when the wings are folded. The pulse frequency rises as the temperature rises. Analysis of the sound oscillograms reveals some differences from the analogous system in cicadas which can be correlated with differences in anatomy. The sinusoidal (inertial) and pulsed modes of operation of insect fibrillar muscle are alternative ways in which oscillatory activity can be generated. They result from a single property of this type of muscle and occur when it is coupled, respectively, to an inertial or to a clicking load.

The physiology of insect fibrillar muscle - IV. The effect of temperature on a beetle flight muscle

The behaviour of an insect fibrillar muscle has been studied over the temperature range 15 to 43°C. The tension/length curves for unstimulated and stimulated muscle are virtually unaffected by changes of temperature. The response of the muscle to sinusoidal changes of length is, however, markedly dependent on temperature. Rise of temperature increases the range of frequencies over which the muscle can do oscillatory work, and also increases the amount of work which it can do per oscillatory cycle. The biological and physico-chemical implications of the results are discussed.

The physiology of insect fibrillar muscle. III. The effect of sinusoidal changes of length on a beetle flight muscle.

The mechanism of the rhythmic activity of insect fibrillar muscle is best investigated by a method which eliminates the effect of load parameters. Because of the peculiar properties of this type of muscle, sinusoidal analysis is more convenient than transient analysis; the validity of this method is established by demonstrating that this muscle has no significant discontinuities in its tension/velocity relationship. Small sinusoidal length changes are im­posed on the muscle with a high-impedance mechanical drive, and the resulting sinusoidal changes of tension give information about the properties of the resting and active muscle. It is shown that the resting muscle can be represented by a visco-elastic model with a time-constant of 1.2 ms. At oscillation frequencies in the range 0 to 55 c/s the stimulated muscle behaves as if there were a time delay of about 7 ms between length changes and tension changes. The muscle has been studied at various lengths, with various frequencies of stimulation and under anoxic conditions. It is not possible to describe its active properties in terms of a simple model; it is suggested instead that an explanation of the effects must be sought in the fine structure of the myofibrils. Oscillatory length changes are not accom­panied by synchronous potential changes in the external surface membrane of the muscle fibres.

The physiology of insect fibrillar muscle - II Mechanical properties of a beetle flight muscle

When an insect fibrillar muscle is stimulated, it may under certain circumstances perform a rhythmical series of contractions at a frequency different from that of the stimuli. This phenomenon has been studied in the basalar muscle of lamellicorn beetles, using specially designed electrical apparatus. The resting muscle is exceedingly stiff. When stimulated, the isometric tension increase is relatively small; active tension/length curves taken under isometric and isotonic conditions are not significantly different. When an inertial load is applied to the muscle, stimulation gives rise to an oscillatory contraction at the mechanical resonant frequency of the system. Although the oscillation frequency is independent of the stimulus frequency, the amplitude of oscillation decreases if the stimulus frequency is reduced below a certain value; there is also a reduction in mean tension. Evidence exists, however, that contraction and oscillatory behaviour are not always so closely associated; different processes may be involved in these two manifestations of muscle activity. Power can be extracted from the muscle by viscous damping of the load, there being an optimum value of damping at which maximum power is obtained. The power output is also dependent on mean muscle tension, reaching a maximum at about the tension which exists in the intact insect. The mechanical properties of the beetle basalar muscle can be correlated with its role in the flight of the insect.

The physiology of insect fibrillar muscle - I. Anatomy and innervation of the basalar muscle of lamellicorn beetles

The anatomy of the basalar muscle and nerve of lamellicorn beetles is described, and a dissection is given for the preparation of the muscle for physiological experiments. The fibres of the muscle are singly innervated, but separate ‘muscle units' are innervated by different nerve fibres, permitting graded action of the muscle as a whole. The neuromuscular junction shows facilitation and conforms to the ‘slow’ arthropod type; muscle potentials increase in amplitude on repetitive stimulation and show little or no overshoot of zero potential. These neuromuscular properties differ from those of previously described insect fibrillar muscles. Electrical activity in the basalar muscle during tethered flight of the whole insect confirms the presence of more than one muscle unit. It is shown that these properties are well adapted to the functioning of a muscle which both provides power for flight and is used in control adjustments.