Effect of active shortening on the rate of ATP utilisation by rabbit psoasmuscle fibres

1. The rate of ATP utilisation during active shortening of single skinned f ibres from rabbit psoas muscle at 10 degreesC was measured using an NADH-li nked assay. Fibres were immersed in silicone oil and illuminated with 365 n m light. The amounts of NADH and carboxytetramethyl-rhodamine (CTMR) in the illuminated region of the fibre were measured simultaneously from fluoresc ence emission at 425-475 and 570-650 nm, respectively. The ratio of these t wo signals was used to determine the intracellular concentration of NADH, a nd thus the ATP utilisation, without interference from movements of the fib re with respect to the measuring light beam. 2. The total extra ATP utilisation due to shortening (Delta ATP) was determ ined by extrapolation of the steady isometric rates before and after shorte ning to the mid-point of the shortening period. Delta ATP had a roughly lin ear dependence on the extent of shortening in the range 1-15 % fibre length (L-0) at a shortening velocity of 0.4 L-0 s(-1) from initial sarcomere len gth 2.7 mum. For shortening of 1% L-0, Delta ATP was 21 +/- 1 muM (mean +/- S.E.M., n = 3). 3. The mean rate of ATP utilisation during ramp shortening of 10% L-0 had a roughly linear dependence on shortening velocity in the range 0.05-1.2 L-0 s(-1). During unloaded shortening at 1.2 L-0 s(-1) the mean rate of ATP ut ilisation was 1.7 mM s(-1), about 9 times the isometric rate. Delta ATP was roughly independent of shortening velocity, and was 84 +/- 9 muM (mean +/- S.E.M., n = 6) for shortening of 10% L-0. 4. The implications of these results for mechanical-chemical coupling in mu scle are discussed. The total ATP utilisation associated with shortening of 1% L-0 is only about 17% of the concentration of the myosin heads in the f ibre, suggesting that during isometric contraction either less than 17% of the myosin heads are attached to actin, or that heads can detach without co mmitment to ATP splitting. The fraction of myosin heads attached to actin d uring unloaded shortening is estimated from the rate of ATP utilisation to be less than 7%.