INFLUENCE OF PHOSPHATE AND PH ON MYOFIBRILLAR ATPASE ACTIVITY AND FORCE IN SKINNED CARDIAC TRABECULAE FROM RAT

1. The effects of inorganic phosphate (P-i) and pH on maximal calcium- activated isometric force and MgATPase activity were studied in chemic ally skinned cardiac trabeculae from rat. ATP hydrolysis was coupled e nzymatically to the breakdown of NADH, and its concentration was deter mined photometrically. Measurements were performed at 2.1 mu m sarcome re length and 20 degrees C. ATPase activity and force were also determ ined when square-wave-shaped length changes were applied, with a frequ ency of 23 Hz and an amplitude of 2.5 %. 2. At pH 7.0 without added P- i, the average isometric force (+/- S.E.M.) was 51 +/- 3 kN m(-2) (n = 23). The average isometric ATPase activity was 0.43 +/- 0.02 mM s(-1) (n= 23). During the changes in length ATPase activity increased to 15 2 +/- 3 % of the isometric value, while the average force level decrea sed to 48 +/- 2 %. 3. Isometric force gradually decreased to 31 +/- 2% of the control value when the P-i concentration was increased to 30 m M. Isometric ATPase activity, however, remained constant for P-i conce ntrations up to 5 mM and decreased to 87 +/- 3 % at 30 mM P-i. When P- i accumulation inside the preparation due to ATP hydrolysis was taken into account, a linear relationship was found between isometric force and log [P-i]. The decrease in relative force was found to be 44 +/- 4 % per decade. 4. During the length changes, ATPase activity and avera ge force showed, apart from the increase in ATPase activity and decrea se in average force, the same dependence on P-i as the isometric value s. Stiffness, estimated from the amplitude of the force responses duri ng the length changes, decreased in proportion to isometric force when the P-i concentration was increased. The changes in the shape of the force responses due to the repetitive changes in length as a function of the P-i concentration were relatively small. These results suggest that the effect of P-i on the transitions which influence ATP turnover is rather insensitive to changes in cross-bridge strain. 5. Isometric force, normalized to the control value at pH 7.0, increased gradually from 54 +/- 1 % pH 6.2 to 143 +/- 10 % at pH 7.5 ATPase activity rema ined practically constant for pH values from 6.8 to 7.2 but decreased to 80 +/- 1 % at pH 6.2 and to 83 +/- 5 % at pH 7.5. ATPase activity d uring the length changes was reduced more than the isometric ATPase ac tivity when pH was lowered. The average force level during length chan ges remained almost constant over the entire pH range. However, the sh ape of the force responses under acidic conditions was pH dependent. T hese observations suggest that at least one of the pH-sensitive transi tions in the cross-bridge cycle which influence ATP turnover is depend ent on changes in cross-bridge strain. 6. To mimic the changes which m ay occur during ischaemia, trabeculae were activated at pH 6.2 in the presence of 30 mM P-i. These experiments showed a decrease in isometri c force and ATPase activity to 14 +/- 1 % and 47 +/- 3 %, respectively . Average force and ATPase activity during the length changes decrease d to 32 +/- 3 % and 55 +/- 3 %, respectively. This indicates that the concurrent changes in [P-i] and pH cause a three- to fourfold increase in isometric tension cost.