EXPERIMENTAL PROTOCOL

Preparations were mounted horizontally to their long axis in the muscle bath, ensuring they were fully immersed in solution and was not contacting the sides of the bath. One tendon was fixed between the clamping forceps and the other tendon attached via a small stainless steel hook to the force transducer. The bathing solution was maintained at 24.0 ± 0.5°C unless otherwise stated. This temperature was chosen in an attempt to minimize the run-down of force seen at higher temperatures (Segal & Faulkner 1985). Isometric contractions were evoked by massive transverse electric field stimulation applied via platinum electrodes. Contraction was thus initiated by a uniform surface membrane action potential. Curare was usually not used. Pulse parameters were

adjusted to ensure supramaximal stimulation for the twitch. Rectangular voltage pulses were set at a duration of 0.5 ms and at 1.1-1.3 X, the amplitude that first produced the maximum twitch force response. The length of the preparation was adjusted to that optimal for twitch force. From a relatively slack length, the fibres were stretched by fine adjustment of a micromanipulator attached to the clamping forceps until there was no further increase in peak twitch force with further stretch. The pulse parameters were checked again before the experiment commenced.

Isometric Contractions

Following equilibration, when stable reproducible twitches and tetani were produced, some basic contractile properties were measured (see Chapter 2.7, Tables 2.2 and 2.3). Fused tetani were induced at a stimulation frequency of 100 Hz in all types of

preparation at 24°C. The frequency required to produce the maximum force in each preparation depended on the muscle fibre-type composition, the presence or absence of innervation, the bathing solution and the temperature. At higher than the optimal frequency, peak tetanic force was often depressed (Chapter 5.3e). The stimulation duration was adjusted to ensure peak tetanic force had been reached. At 100 Hz the duration was usually 1 s in normal and denervated soleus fibres and 250-300 ms in stemomastoid fibres. In fatigue studies, the preparations were tetanically stimulated for a duration in which force was reduced to less than 50% of peak tetanic force. This ranged from 1-2 s in stemomastoid fibres to 30 s in denervated soleus fibres.

19

Preparations that displayed a rapid run-down of peak tetanic force, i.e. > 0.5% per min, or large force undershoots following tetani were regarded as being unhealthy and were rejected.

Potassium Contractures

K-contractures were induced by rapidly exposing the preparation to solutions containing high [K+] (60K or 200K, Table 2.1). These solutions were applied by gravitational flow via polystyrene tubing at a maximal rate of 2 ml/s. A high [K+] solution was applied until contracture force had just started to decline from its peak (i.e. at the start of mechanical inactivation). It was then washed out with the control Solution 1. The contracture was not due to the higher ionic strength of 10K, 60K, 200K than Solution 1 since all high [K+] solutions had the same ionic strength and 10K did not produce a contracture. K-contracture amplitude was normalized to peak tetanic force to eliminate variations due to preparation size. Following wash-out of high [K+] solutions, no further contractures were induced until peak tetanic force had recovered to a steady force level close to the pre-contracture force. Normalized K-contracture force was usually reproducible to within 5% for a given preparation. 200K normally produces maximal contractures in normal and denervated soleus fibres (Chua & Dulhunty 1988). In the present study the 200K-contracture amplitude was close to or greater than peak tetanic force. However, it was important that at least one diameter of the preparation was only one or two fibres thick to achieve this ratio.

Drug Administration

Drugs were usually added to solutions just prior to commencement of experiments. Drugs were administered to the fibres by bath application using either (i) gravitational flow as for K-contractures, via the water jacket or directly into the muscle bath, or (ii) glass micropipettes when rapid exposure to drugs was not required.

Concentration-response relations were tested in two ways. Drugs that did not readily wash-out or caused tachyphylaxis (i.e. smaller responses on successive applications) were added cumulatively whereas other drugs were added in a random order.

Run-down

A problem with mechanical studies in isolated mammalian preparations is that of a run-down of force with time. Run-down was the fastest in the stemomastoid, slower in the normal soleus and the slowest in the denervated soleus preparations. It is unlikely that run-down was due to the presence of physically damaged fibres or the loss of individual fibres as discrete steps of force loss would have been expected, especially in the smaller bundles, but this was not seen. Preparation thickness, bubbling the solution

20

with 100% C>2 and a temperature of 24°C were all optimised to reduce force loss due to hypoxia (Goldberg et al. 1975; Segal & Faulkner 1985). The ratio of 200K-contracture force to peak tetanic force stayed much the same during run-down indicating that the action potential is not impaired. Run-down may be related to the use-dependent decline in voltage activated force seen in skinned fibres and attributed to an impairment of E-C coupling in vitro (Lamb & Stephenson 1990a).