Ex vivo EDL muscle contractility measurements

Ex vivo assessment of muscle force production was made in intact, excised EDL muscles. Mice were anaesthetized by intra-peritoneal injection of anaesthetic (50). EDL muscles were isolated, tied using 4–0 surgical suture, excised and then attached to a servo motor and force transducer (Aurora Scientific, Ontario, CA) between two platinum electrode plates in a chamber continuously perfused with oxygenated Ringer’s solution. Optimal stimulation level and muscle length (L₀) were determined using a series of 1 Hz twitch stimulation trains while stretching the muscle to a length that generated maximal force (F₀). After establishing L₀, muscles were first equilibrated using three tetani (500 ms, 150 Hz) given at 1 min intervals. After 3 min of additional equilibration, muscles were subjected to a standard force frequency protocol (from 1 to 250 Hz). All muscle contractility experiments were carried out at 30°C. Muscle force was recorded using Dynamic Muscle Control software (Aurora Scientific, Ontario, CA) and analysed using a combination of both Dynamic Muscle Analysis (Aurora Scientific, Ontario, CA) and Clampfit 10.0 (Molecular Devices, San Jose, CA) software. Specific force was calculated by normalizing the absolute force to the physiological cross sectional area as previously described (51,52).