Effect of active shortening on the rate of ATP utilisation by rabbit psoas muscle fibres.

Abstract

1. The rate of ATP utilisation during active shortening of single skinned fibres from rabbit psoas muscle at 10 degrees C was measured using an NADH-linked assay. Fibres were immersed in silicone oil and illuminated with 365 nm light. The amounts of NADH and carboxytetramethylrhodamine (CTMR) in the illuminated region of the fibre were measured simultaneously from fluorescence emission at 425-475 and 570-650 nm, respectively. The ratio of these two signals was used to determine the intracellular concentration of NADH, and thus the ATP utilisation, without interference from movements of the fibre with respect to the measuring light beam. 2. The total extra ATP utilisation due to shortening (ATP) was determined by extrapolation of the steady isometric rates before and after shortening to the mid-point of the shortening period. ATP had a roughly linear dependence on the extent of shortening in the range 1-15 % fibre length (L0) at a shortening velocity of 0.4 L0 s-1 from initial sarcomere length 2.7 microm. For shortening of 1 % L0, ATP was 21 +/- 1 M (mean +/- S.E.M., n = 3). 3. The mean rate of ATP utilisation during ramp shortening of 10 % L0 had a roughly linear dependence on shortening velocity in the range 0.05-1.2 L0 s-1. During unloaded shortening at 1.2 L0 s-1 the mean rate of ATP utilisation was 1.7 mM s-1, about 9 times the isometric rate. ATP was roughly independent of shortening velocity, and was 84 +/- 9 microM (mean +/- S.E.M., n = 6) for shortening of 10 % L0. 4. The implications of these results for mechanical-chemical coupling in muscle are discussed. The total ATP utilisation associated with shortening of 1 % L0 is only about 17 % of the concentration of the myosin heads in the fibre, suggesting that during isometric contraction either less than 17 % of the myosin heads are attached to actin, or that heads can detach without commitment to ATP splitting. The fraction of myosin heads attached to actin during unloaded shortening is estimated from the rate of ATP utilisation to be less than 7 %.