Ca-dependence of diastolic properties of cardiac sarcomeres: involvement of titin |
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Authors: | B D Stuyvers M Miura J-P Jin H E D J ter Keurs |
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Institution: | a Medical Physiology, University of Calgary Calgary, Alberta Canada b Department of Physiology and Biophysics, Case Western Reserve University Cleveland, OH USA |
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Abstract: | The stiffness of the sarcomeres was studied during the diastolic interval of 18 stimulated (0.5 Hz) cardiac trabeculae of rat (pH 7.4; temperature = 25°C). Sarcomere length (SL) and force (F) were measured using, respectively, laser diffraction techniques (resolution: 4 nm) and a silicon strain gauge (resolution: 0.63 μN). Sinusoidal perturbations (frequency = 500 Hz) were imposed to the length of the preparation. The stiffness was evaluated from the corresponding F and SL sinusoids by analysis of both signals together either in the time domain or in the frequency domain. A short burst (duration = 30 ms) of sinusoidal perturbations was repeated at 5 predetermined times during diastole providing 5 measurements of stiffness during the time interval separating two twitches. These measurements revealed that stiffness increases by 30% during diastole, while a simultaneous expansion of the sarcomeres (amplitude = 10-60 nm) was detected. Measurements of the fluorescence of fura-2 under the same conditions revealed a continuous exponential decline of Ca2+]i from 210 to 90 nM (constant of time 300 ms) during diastole. In order to test the possibility 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 430 nM on sarcomere stiffness in rat cardiac trabeculae skinned by saponin (n = 17). Stiffness was studied using 500 Hz sinusoidal perturbations of muscle length (ML). We found that, below 70 nM, the stiffness was independent of Ca2+]; between 70 and 200 nM, the stiffness declined with increase of Ca2+]; above 200 nM, the stiffness increased steeply 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 μM; n = 2.1±0.2) due to attachment of cross-bridges. From these results, it was possible to reproduce accurately the time course of diastolic stiffness observed in intact trabeculae and to predict the effect on stiffness of a spontaneous elevation of the diastolic Ca2+]. Identical stiffness 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 μM) eliminated the Ca2+-dependence of stiffness. These results are consistent with the hypothesis that the Ca2+-sensitivity of the sarcomeres at Ca2+] < 200 nM, i.e. where the myocytes in intact muscle operate during diastole, involves an association between titin molecules and the thin filament. |
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Keywords: | Ca2+ Sarcomere Visco-elasticity Stiffness Titin |
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