Ca(2+)-dependence of passive properties of cardiac sarcomeres.

Rat cardiac trabeculae constitute a well-known experimental model in studies of cardiac contraction at the sarcomere level. Continuous measurement of length of the sarcomeres (SL) by laser diffraction technique permits one to monitor the active shortening of the contractile units during generation of force. When the preparation is stimulated repetitively (0.5 Hz) by electrical pulses, active shortenings are separated by periods corresponding approximately to the diastolic interval in the heart and wherein normally no major contractile event would have been expected. In contrast to this expectation, studies conducted with high-resolution (2-4 nm) SL measurements technique revealed that sarcomeres continuously lengthened (by 10-60 nm) from the end of twitch relaxation to the next stimulation. Such lengthening resulted from an internal expansion of the sarcomere and not from stretch exerted by extra-sarcomeric sources. We further characterized diastolic changes by measuring sarcomere stiffness (Sarc-Stiff) estimated from the response to short bursts (30 ms) of sinusoidal perturbations (frequency: 500 Hz) at 5 moments of the resting interval separating twitches. Sarc-Stiff increased continuously by approximately 30% during the diastolic interval (29 degrees C, pH: 7.4, [Ca2+]o = 1 mM). We then investigated during the same period the intracellular dynamics of Ca2+, as a major determinant of sarcomere motions in muscle. Intracellular free-Ca2+ concentration ([Ca2+]i) was measured continuously in trabeculae microinjected with the fluorescent Ca(2+)-probe Fura-2 and stimulated at 0.5 Hz. It appeared that the Ca(2+)-transient, which drives the twitch, did not end with the apparent relaxation of the force. Instead, [Ca2+]i kept decreasing in an exponential manner throughout the diastolic interval. At [Ca2+]o = 1 mM, [Ca2+]i decreased from 230 to 90 nM with a time constant of approximately 250 ms. The similarity in time courses of Ca(2+)-decline and of Sarc-Stiff increase suggested that properties of resting sarcomeres were related to [Ca2+]i in the sub-micromolar range. In order to examine this possibility, Sarc-Stiff was measured in chemically skinned trabeculae, i.e. in a preparation allowing control of [Ca2+] surrounding the sarcomeres. Sarc-Stiff was measured at different [Ca2+] from 1 to 450 nM. We found that 1) below 70 nM, Sarc-Stiff was independent on [Ca2+], 2) between 70 and 200 nM, i.e., approximately the range wherein [Ca2+]i decreased during diastole in intact muscle, Sarc-Stiff decreased by approximately 50% with increase of [Ca2+] and 3) above 200 nM, Sarc-Stiff increased steeply with increase of [Ca2+] as was expected from Ca(2+)-dependent attachment of cross-bridges between actin and myosin. The data fitted accurately to the sum of 2 sigmoid functions: 1) at [Ca2+] < 200 nM, Sarc-Stiff decreased with increase of [Ca2+] with a Hill coefficient (nH) = -2.6 and [Ca2+] at half maximal activation (EC50) = 0.16 +/- 0.013 microM; 2) at [Ca2+] > 200 nM, Sarc-Stiff increased with [Ca2+] (nH: 2.1; EC50:3.4 +/- 0.3 microM) consistent with Ca(2+)-dependent attachment of cross-bridges. It was possible to reproduce the diastolic variation of Sarc-Stiff observed in intact muscle by using the time course of [Ca2+]i in the Sarc-Stiff--[Ca2+] relationship determined from skinned trabeculae. We conclude that physical properties of the sarcomeres are inversely related to Ca2+ below 200 nM, i.e., in a range of concentrations where the myocytes operate during diastole while the influence of cross-bridges is negligible.

Ca(2+)-dependence of diastolic properties of cardiac sarcomeres: involvement of titin.

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 degrees 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 microN). 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 approximately 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 approximately 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 Ca(2+)-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 microM; 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 microM) eliminated the Ca(2+)-dependence of stiffness. These results are consistent with the hypothesis that the Ca(2+)-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.

Dynamics of viscoelastic properties of rat cardiac sarcomeres during the diastolic interval: involvement of Ca2+.

1. Cardiac sarcomere stiffness was investigated during diastole in eighteen trabeculae dissected from the right ventricle of rat heart. The trabeculae were stimulated at 0.5 Hz, in a modified Krebs-Henseleit solution (pH, 7.4; 25 degrees 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 approximately 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 nM external Ca2+ concentration ([Ca2+]o), the average stiffness modulus (Mod) increased from 9.3 +/- 0.6 to 12 +/- 0.6 nN mm-2 micron-1 (n = 18; P < 0.05), while the average phase shift (phi) 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 phi 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.

Diastolic viscoelastic properties of rat cardiac muscle; involvement of Ca2+.

Diastolic cardiac sarcomere stiffness, sarcomere length changes, and calcium concentration [Ca2+]i were investigated in 18 trabeculae, dissected from the right ventricle of rat heart. [Ca2+]i declined following a mono-exponential diastolic time course with a time constant of 210-350 ms. During diastole, ([Ca2+]o = 1 mM); sarcomere length (SL) increases (amplitude: 5-65 nm; time constant: 600 ms). Eighty percent of muscles showed discrete spontaneous motion of sarcomeres near the end of diastole; this phenomenon occurred earlier at higher [Ca2+]o. The stiffness modulus of the sarcomere (MOD) increased by 30% during diastole (n = 158; p < 0.05), while the phase difference, phi, between force and SL decreased by 13% (n = 158; p < 0.05). The increase of MOD and the decrease of phi reversed when spontaneous activation occurred. These results show that the mechanical diastolic properties of the cardiac sarcomere are time dependent. The time dependence of the diastolic properties can be faithfully reproduced by a simple linear four element viscoelastic model. The diastolic changes of MOD and of phi could be reproduced by assuming an exponential change of the elastic and viscous coefficients of the model over time with a time constant similar to the time constant of change of [Ca2+]i. We suggest that the simplest combination of structural counterparts of the model in the sarcomere consists of titin bound to both actin and myosin in the myofibril, while the sarcomere is in parallel with another purely elastic element. We propose that the Ca(2+)-dependence of diastolic stiffness might be the result of an inverse relation between [Ca2+]i and the affinity of titin for actin.