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.

Effect of angiotensin II on calcium release phenomena in normal and hypertrophied single cardiac myocytes.

The effects of angiotensin II (Ang II) (10(-9) M to 10(-7) M) on calcium releases were established in ventricular myocytes from normal and renal hypertensive adult rats. From each peak systolic indo-1 ratio (405 nm/480 nm), amplitude variation, duration (rise time and fall time), and frequency of spontaneous calcium releases were investigated on freshly isolated cardiomyocytes at rest or under electrical stimulation. The following changes were observed: (1) in spontaneous contracting myocytes, an increase in frequency of calcium transients at 10(-7) M in normal cells (+157%, P < 0.05) and at whatever angiotensin II concentration in hypertrophied cells (10(-9) M: +79% P < 0.05; 10(-8) M +82%, P < 0.01; 10(-7) M: +285%, P < 0.01) with a greater sensitivity of hypertrophied cells to Ang II (P < 0.05 at 10(-9) M, P < 0.01 at 10(-8) M). (2) In stimulated myocytes, a prolongation of the duration of calcium transients at 10(-7) M in normal cells (+68%, P < 0.01) and at 10(-9) M, 10(-8) M, 10(-7) M in hypertrophied cells: (+36%, P < 0.05; +39%, P < 0.01; +77%, P < 0.01) with a greater sensitivity of hypertrophied myocytes (P < 0.05 at 10(-9) M and 10(-8) M). An increase in duration may be explained by the occurrence of calcium releases during the fall time of calcium transients. Thus, both in normal and hypertrophied myocytes, Ang II induced the occurrence of calcium releases with increased sensitivity of hypertrophied cells to Ang II. Such calcium releases are known to be a possible cause of arrhythmias termed "triggered activity".

[Effects of angiotensin II on spontaneous cytosolic calcium releases in ventricular myocytes of adult normal and hypertensive rats]. / Effets de l'angiotensine II sur les libérations spontanées calciques cytosoliques dans les myocytes ventriculaires isolés de rats adultes normaux et hypertendus.

This study investigated the effects of angiotensin II (Ang II) (10(-9)M to 10(-7)M) on calcium releases in ventricular myocytes from normal and renal hypertensive adult rats (Goldblatt 2K-1C). Newly, isolated myocytes were loaded with fluorescent indo-1/AM and studied at rest or under electrical stimulation. The variation of the ratio of indo-1 emission (405 nm/480 nm) was taken as a measure of cytosolic calcium variations. Five parameters were investigated from each peak systolic indo-1 ratio before and after Ang II addition: amplitude variation, duration with analysis of a rise time and a fall time, and frequency of spontaneous calcium releases. Following changes were observed: in unstimulated myocytes exhibiting spontaneous contractile activity, increase in frequency of calcium transients, at 10(-7)M, in normal cells (+ 157 +/- 27%; p < 0.01) and whatever Ang II concentration in hypertrophied cells (+ 79 +/- 31%; p < 0.01; + 82 +/- 25%, p < 0.01; + 285 +/- 50%, p < 0.01 à 10(-9)M, 10(-8)M, 10(-7)M); in stimulated myocytes, prolongation of the duration of calcium transients explained by the occurrence of calcium releases during fall time. In addition, 50% of myocytes exhibited spontaneous releases of calcium in the interstimulus interval. Increase in calcium transients duration was statistically significant, whatever Ang II concentration in hypertrophied cells (+ 36 +/- 20%, p < 0.05; + 39 +/- 18%, p < 0.01; + 77 +/- 34%, p < 0.01 à 10(-9)M, 10(-8)M, 10(-7)M) and only at 10(-7)M in normal cells (+ 68 +/- 22% p < 0.01). Similar results were observed in fall time.(ABSTRACT TRUNCATED AT 250 WORDS)

Nicardipine and cardiac hypertrophy: effects on left ventricular mass. Haemodynamics and mechanical performance in renovascular hypertensive rats.

SUBJECT OBJECTIVE: The aim was to determine the effects of nicardipine treatment (10-15 mg.kg-1.d-1 intraperitoneal) on left ventricular hypertrophy, coronary haemodynamics, and mechanical performance in renovascular hypertensive rats. DESIGN: Systemic and coronary haemodynamic variables were evaluated by using microspheres in conscious rats, while mechanical performance and elasticity were measured on isolated papillary muscles from the same animals. EXPERIMENTAL ANIMALS: Male Sprague-Dawley rats, weight 150-180 g, were used. Nine treated rats were compared with control groups, consisting of 9 sham operated, 12 untreated hypertensive, and 9 nephrectomised rats. MEASUREMENTS AND MAIN RESULTS: Eight weeks after clipping, removal of the ischaemic kidney allowed the normalisation of ventricular mass and the reversal of changes induced by hypertrophy except prolongation of timing parameters. Nicardipine administration led to an efficient but incomplete control of blood pressure, which fell from 208(SEM 5) mm Hg in untreated hypertensive rats to 155(4) mm Hg in treated rats, p less than 0.01. After eight weeks of treatment, the reduction in left ventricular mass, from 3.10(0.10) mg.g-1 in untreated rats to 2.72(0.018) mg.g-1 in treated rats was significant (p less than 0.01) but was less than the pressure decrease. In treated hypertensive rats, coronary blood flow was increased and redistributed at rest in favour of the subepicardial layers but was significantly reduced after maximum vasodilatation, to 1.618(0.077) litre.min-1.100 g, in nicardipine treated rats. A reversal of impaired myocardial mechanical indices towards control values was observed except for time to peak force and time to half relaxation. CONCLUSIONS: Nicardipine treatment allowed blood pressure control, reduction of left ventricular mass, and improvement in mechanical performance, but was unable to restore minimal coronary vascular resistance. The results suggest that, although blood pressure decrease is obviously important, it may not be the sole factor in the reversal of cardiac hypertrophy.

Digital-imaging microscopy analysis of calcium release from sarcoplasmic reticulum in single rat cardiac myocytes.

Digital imaging microscopy of fura-2 fluorescence has allowed us to assess the dynamic patterns of local Ca increase in newly isolated rat myocardial cells. Of the myocytes bathed in a saline solution (1.8 mM Ca2+, 37 degrees C, pH 7.4), 10%-20% exhibited local spontaneous contractions. The resting intracellular free calcium concentration ([Ca2+]i) of these cells was 106 +/- 4 nM versus 77 +/- 3 nM for non-contracting cells. The spontaneous contractile activity appeared to be closely related to internal spontaneous Ca waves that spread across the myoplasm (velocity approximately 50 microns/s, maximal Ca amplitude = 195 +/- 11 nM) along the major axis of the cells. Precise topographical examination of Ca wave propagation indicated a refractory period for internal Ca release. The occurrence of both the generation and propagation of spontaneous Ca increases appeared to be closely dependent on the extent of Ca loading of the cells. Most of our observations were in accordance with the assumption that local Ca overload of the sarcoplasmic reticulum (SR) is the main parameter involved in the spontaneous Ca-release phenomena. Using the same approach, the increase in internal Ca evoked by KCl (50 mM) addition was investigated, and compared with that seen during spontaneous activity. Total [Ca2+]i increase induced by K+ depolarization involved three consecutive local Ca-release patterns: (a) a peripheral Ca enhancement that remained during the total [Ca2+]i increase, (b) subsequent transversal local Ca increases occurring in Z-line regions, (c) longitudinal local Ca increases. In addition, a weak heterogeneous Ca distribution was detected in both peripheral and central parts of resting cardiac cells. Thus, the total Ca increase seemed to result consecutively from a peripheral Ca pool, from junctional SR and from longitudinal structures (possibly longitudinal SR).

[Effects of ventricular hypertrophy following experimental renovascular hypertension on the elasticity of the papillary muscle]. / Incidence d'une hypertrophie ventriculaire consécutive à une hypertension rénovasculaire expérimentale sur l'élasticité du muscle papillaire.

A precise method was developed for detecting muscular stiffness changes in the left ventricular hypertrophy by chronic pressure overload in rats (2 kidney-1 clip renal hypertensive rats). The technique of Quick-Release allowed the measurement of the "stress-strain" relation (delta-epsilon), the elastic stiffness (d delta/d epsilon) and the stiffness constant (K) in isolated papillary muscle from normal and hypertrophied hearts at rest and at the maximal time course of activation during isometric contraction. (Table: see text). The results of this study reported that the cardiac hypertrophy induced by experimental pressure overload was not associated with significant alteration of myocardial stiffness. So, it would seem that a muscular stiffness modification was unresponsible for the ventricular compliance change of clinical and experimental results.

[Effects of nicardipine on left ventricular hypertrophy of the rat with renovascular arterial hypertension]. / Effets de la nicardipine sur l'hypertrophie ventriculaire gauche du rat porteur d'une hypertension artérielle rénovasculaire.

This study investigate the effects of Nicardipine treatment on regression of left ventricular hypertrophy (LVH), coronary hemodynamic and myocardial mechanical performance. 30 Sprague-Dawley male rats were divided into 3 groups: sham operated rats control group (SHC), untreated hypertensive rats group (RHR-U), treated hypertensive rats group (RHR-N). Systemic and coronary hemodynamics were determined by using left atrial injection of radioactive microspheres, 16 weeks after clipping. Mechanical performance was measured on isolated papillary muscle from the same animal. Results (mean +/- SEM) (Table: see text). Nicardipine treatment (10 to 15 mg intraperitoneal dosage during 8 weeks), led to: an efficient but incomplete control of hypertension. a reduction of left ventricular mass in proportion lesser than pressure decrease. a raise of coronary blood flow at rest with inversion of flow distribution between endocardium an epicardium. a decrease of "maximal" coronary blood flow. a reversal of impaired myocardial mechanical parameters towards control values except for contraction timing parameters. Decrease of "maximal" coronary blood flow could have deleterious effects on cardiac function.