Mechanical measurements from single frog skeletal muscle myofibrils activated by rapid solution changes

Single myofibrils isolated from striated muscle represent a viable experimental model for the study of the mechanics of muscle contraction (Bartoo et al., J. Muscle Res. Cell Motil. 14, 498–510, 1993; Friedman & Goldman, Biophys. J. 64, 345a, 1993). With this preparation of extremely small cross-sectional area it is possible to obtain a rapid diffusional equilibrium following changes of the bathing medium. In this study we present a novel experimental method which allows solution changes within 50 ms resulting in rapid increase in calcium concentration in the close environment of the specimen and/or in application of rapid perturbation of the composition of the experimental solution. Experiments were performed using single myofibrils or thin bundles of 2–3 myofibrils, 50–100 ìm long, prepared by homogenization of frog glycerinated tibialis anterior muscle. The preparations were mounted horizontally between the lever arms of an isometric force transducer and a length control motor (Colomo et al., J. Physiol. 475, 347–50, 1994) in a temperature controlled trough filled with relaxing solution (pCa 8, 3 mM MgATP; 158 C). Sarcomere length was set just above slack length (2.1–2.2 ìm). Mounted myofibrils were continuously perfused by one of two parallel streams of solution jetted by gravity from a theta style glass capillary positioned at right angle with the experimental preparation and at a distance of 500 ìm. Each pipette channel (200 ìm tip diameter) was connected to reservoirs filled with either relaxing or activating (pCa 4.75) solution. The perfusion system was firmly attached to a stepping motor that could be operated to cause rapid alternation of the stream flowed over the myofibril. The mechanical artefact associated with the movement of the perfusion pipette lasted 100–150 ms. When myofibrils were subjected to cycles of activation and relaxation using this experimental device and at high ATP concentration (3 mM), both tension rise and tension relaxation were complete before the end of the mechanical artefact. The maximal activated force of single myofibrils averaged 0.45 0.03 ìN (nˆ16) which corresponds to a mean tension of 376 21 kNmÿ2. Myofibrils activated at low MgATP concentration (60 ìM) showed a markedly slower time course of tension development. Preliminary results on single myofibrils activated and then subjected to a rapid decrease in MgATP concentration indicate a moderate increase in the level of isometric force developed at the contracture plateau, in agreement 180 Abstracts with previous results on whole skinned fibres (Ferenczi et al., J. Physiol. 350, 519–43, 1984).