Oscillatory contraction of insect fibrillar muscle after glycerol extraction

Abstract

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.