CA2-DEPENDENCE OF DIASTOLIC PROPERTIES OF CARDIAC SARCOMERES - INVOLVEMENT OF TITAN()

The stiffness of the sarcomeres was studied during the diastolic inter val of 18 stimulated (0.5 Hz) cardiac trabeculae of rat (pH 7.4; tempe rature = 25 degrees C). Sarcomere length (SL) and force (F) were measu red using, respectively, laser diffraction techniques (resolution: 4 n m) and a silicon strain gauge (resolution: 0.63 mu N). Sinusoidal pert urbations (frequency = 500 Hz) were imposed to the length of the prepa ration. The stiffness was evaluated from the corresponding F and SL si nusoids by analysis of both signals together either in the time domain or in the frequency domain. A short burst (duration = 30 ms) of sinus oidal perturbations was repeated at 5 predetermined times during diast ole providing 5 measurements of stiffness during the time interval sep arating two twitches. These measurements revealed that stiffness incre ases by similar to 30% during diastole, while a simultaneous expansion of the sarcomeres (amplitude = 10-60 nm) was detected. Measurements o f the fluorescence of fura-2 under the same conditions revealed a cont inuous exponential decline of [Ca2+](i) from 210 to 90 nM (constant of time similar to 300 ms) during diastole. In order to test the possibi lity that the increase of sarcomere stiffness and the decline of [Ca2](i) were coupled during diastole of intact trabeculae, we studied the effect of different free Ca2+-concentrations ([Ca2+]) between 1 and 4 30 nM on sarcomere stiffness in rat cardiac trabeculae skinned by sapo nin (n = 17). Stiffness was studied using 500 Hz sinusoidal perturbati ons of muscle length (ML). We found that, below 70 nM, the stiffness w as independent of [Ca2+]; between 70 and 200 nM, the stiffness decline d with increase of [Ca2+]; above 200 nM, the stiffness increased steep ly with [Ca2+]. The data fitted accurately to the sum of two sigmoids (Hill functions): (1) at [Ca2+] < 200 nM the stiffness decreased with [Ca2+] (EC50 = 160 +/- 13 nM; n = 2.6 +/- 0.7) and (2) at [Ca2+] > 200 nM, stiffness increased with [Ca2+] (EC50 = 3.4 +/- 0.3 mu M; n = 2.1 +/- 0.2) due to attachment of cross-bridges. From these results, it w as possible to reproduce accurately the time course of diastolic stiff ness observed in intact trabeculae and to predict the effect on stiffn ess of a spontaneous elevation of the diastolic [Ca2+]. Identical stif fness measurements were performed in 4 skinned preparations exposed to a cloned fragment of titin (Ti I-II) which has been shown to exhibit a strong interaction with F-actin in vitro. It was anticipated that Ti I-II would compete with endogenous titin for the same binding site on actin in the I-band. Below 200 nM, Ti I-II (2 mu M) eliminated the Ca 2+-dependence of stiffness. These results are consistent with the hypo thesis that the Ca2+-sensitivity of the sarcomeres at [Ca2+] < 200 nM, i.e. where the myocytes in intact muscle operate during diastole, inv olves an association between titin molecules and the thin filament. (C ) 1998 Elsevier Science Ltd. All rights reserved.