1. Cardiac sarcomere stiffness was investigated during diastole in eighteen trabeculae dissected from the right ventricule of rat heart. The trabeculae were stimulated at 0.5 Hz, in a modified Krebs-Henseleit solution (pH, 7.4; 25°C). Sarcomere length (SL) was measured using high resolution (± 2 nm) laser diffraction techniques. Force (F) was measured with a silicon strain gauge. 2. SL increased exponentially (amplitude, 25 ± 9 nm; n = 15) throughout diastole. This increase occurred even at slack SL, showing that this phenomenon was due to an internal expansion. The majority of the muscles showed discrete spontaneous fluctuations of SL (amplitude < 20 nm) starting ~ 1 s after the end of the twitch. 3. The intracellular free Ca2+ concentration ([Ca2+](i)) was measured from the fluorescence of microinjected fura-2 salt in seven trabeculae under the same experimental conditions. [Ca2+](i) continuously declined (from 240 to 90 nM) during diastole following a monoexponential time course (time constant, 210-325 ms). 4. The stiffness of the sarcomere was evaluated at 10, 30, 50, 70 and 90% of diastole using bursts (30 ms) of 500 Hz sinusoidal perturbations of muscle length (amplitude of SL oscillations < 30 nm). At 1 mM external Ca2+ concentration ([Ca2+](o)), the average stiffness modulus (Mod) increased from 9.3 ± 0.6 to 12 ± 0.6 mN mm-2 μm-1 (n = 18; P < 0.05), while the average phase shift (Φ) between F and SL signals decreased from 84 ± 3 to 73 ± 4 deg (n = 18; P < 0.05) between 10 and 90% during diastole. The increase in Mod and the decrease in Φ reversed when spontaneous activity occurred. When [Ca2+](o) was raised to 2 mM, the stiffness time course reversed approximately 450 ms earlier, simultaneously with the occurrence of spontaneous activity. 5. Our results show that diastole is only an apparent steady state and suggest that the structural system responsible for the viscoelastic properties of the sarcomere is regulated by [Ca2+](i) in the submicromolar range. Different possible origins of the dynamic changes in viscoelasticity during diastole are discussed.
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