Inhibitors of SERCA and mitochondrial Ca-uniporter decrease velocity of calcium waves in rat cardiomyocytes.

Spontaneous calcium waves in isolated rat cardiomyocytes were investigated by confocal laser scanning microscopy using the fluorescent Ca(2+)-indicator fluo-4 AM. With increasing calcium overload propagation velocities reinforced. The calcium wavespeed was significantly diminished by drugs which interfere with the calcium uptake of both the sarcoplasmic reticulum (SR) and mitochondria, respectively. Stepwise addition of thapsigargin, a highly specific inhibitor of SERCA, decreased the wavespeed and allowed the determination of flux control coefficients which were found to be increasing from 0.15-0.75 in dependence on calcium overload. Kd was estimated to be between 0.4 and 0.6 nM TG. At 5 mM TG wavespeed was significantly reduced by almost 50%. Spontaneous calcium waves did not occur in bathing solutions with more than 20 nM thapsigargin. Calcium wave velocity was also reduced in the presence of the oxygen-bridged dinuclear ruthenium amine complex RU 360 which specifically blocks the mitochondrial Ca2+ uptake. The observed effects are likely due to a reduction of the ryanodine receptor's open probability. It is suggested that the intracellular Ca2+ signaling depends on both SR lumenal and cytosolic calcium concentration.

Calcium waves in agarose gel with cell organelles: implications of the velocity curvature relationship.

Calcium oscillations and waves have been observed not only in several types of living cells but also in less complex systems of isolated cell organelles. Here we report the determination of apparent Ca2+ diffusion coefficients in a novel excitable medium of agarose gel with homogeneously distributed vesicles of skeletal sarcoplasmic reticulum. Spatiotemporal calcium patterns were visualized by confocal laser scanning fluorescence microscopy. To obtain characteristic parameters of the velocity curvature relationship, namely, apparent diffusion coefficient, velocity of plane calcium waves, and critical radius, positively and negatively curved wave fronts were analyzed. It is demonstrated that gel-immobilized cell organelles reveal features of an excitable medium. Apparent Ca2+ diffusion coefficients of the in vitro system, both in the absence or in the presence of mitochondria, were found to be higher than in cardiac myocytes and lower than in unbuffered agarose gel. Plane calcium waves propagated markedly slower in the in vitro system than in rat cardiac myocytes. Whereas mitochondria significantly reduced the apparent Ca2+ diffusion coefficient of the in vitro system, propagation velocity and critical size of calcium waves were found to be nearly unchanged. These results suggest that calcium wave propagation depends on the kinetics of calcium release rather than on diffusion.

Sarcomere length-tension relationship of rat cardiac myocytes at lengths greater than optimum.

The study was aimed at determining both passive and Ca(2+)-activated forces of single skinned rat cardiac cells. Particular attention was paid to the descending limb of the active length-tension curve while the sarcomeric order of stretched cells was investigated before and during contraction. To analyse sarcomere length and sarcomere-length inhomogeneity, a fast Fourier transform (FFT) was employed. The fundamental frequency in the FFT spectrum is a measure of sarcomere length. The full-width-half-maximum of the first-order line is a measure of sarcomere-length inhomogeneity. In relaxing buffer, the sarcomere-length inhomogeneity of skinned cells increased linearly with mean sarcomere length. Upon Ca(2+)-dependent activation of skinned cells contracting isometrically, mean sarcomere length decreased slightly and inhomogeneity increased; both effects were greater at higher Ca(2+)concentrations. Maximum activation was reached at sarcomere lengths between 2.2 and 2.4 microm, whereas the descending limb of the active length-tension curve approached zero force already at approximately 2.8 microm. This steep force decline could not be explained by overly inhomogeneous sarcomere lengths in very long, contracting cells. Rather, the results of mechanical measurements on single cardiac myofibrils implied that high stretching is accompanied by irreversible structural alterations within cardiac sarcomeres, most likely thick-filament disarray and disruption of binding sites between myosin and titin due to changes in titin's tertiary structure. Loss of a regular thick-filament organization may then impair active force generation. We conclude that the descending limb of the cardiac length-tension curve is determined both by the degree of actin-myosin overlap and by the intrinsic properties of titin filaments.

Sarcoplasmic reticulum vesicles embedded in agarose gel exhibit propagating calcium waves.

In different cell types, activation of signal transduction pathways leads to the generation of calcium oscillations and/or waves. Due to this important impact for cellular function, calcium waves are the subject of intensive investigations. To study interactions of cell organelles with no influence of the cell membrane, sarcoplasmic reticulum (SR) vesicles and well-coupled mitochondria were reconstituted. For the first time, we demonstrate the generation and propagation of calcium waves in a suspension of sarcoplasmic reticulum vesicles, embedded in an agarose gel. The propagation dynamics resemble those of calcium waves in living cells. Moreover, the addition of well-coupled mitochondria leads to more pronounced and significantly faster propagating waves, demonstrating the importance of the mitochondrial Ca(2+) transport. The experimental and simulation results indicate the resemblance of the in vitro system to an excitable medium.

Striational autoantibodies in myasthenia gravis patients recognize I-band titin epitopes.

Myasthenia gravis (MG) patients develop autoantibodies primarily against the acetylcholine receptor in the motor endplate, but also against intracellular striated muscle proteins, notably titin, the giant elastic protein of the myofibrillar cytoskeleton. Titin antibodies have previously been shown to be directed against a single epitope on the molecule, located at the A-band/I-band junction and referred to as the main immunogenic region (MIR) of titin. By using immunofluorescence microscopy on stretched single myofibrils, we now report that approximately 40% of the sera from 18 MG/thymoma patients and 8 late-onset MG patients with thymus atrophy contain antibodies that bind to a more central I-band titin region. This region consists of homologous immunoglobulin domains and is known to be differentially spliced dependent on muscle type. All patients with I-band titin antibodies also had antibodies against the MIR. Although a statistically significant correlation between the occurrence of I-band titin antibodies and MG severity was not apparent, the results could hint at an initial immunoreactivity to titin's MIR, followed by reactivity along the titin molecule in the course of the disease.

Velocity-curvature relationship of colliding spherical calcium waves in rat cardiac myocytes.

Colliding spherical calcium waves in enzymatically isolated rat cardiac myocytes develop new wavefronts propagating perpendicular to the original direction. When investigated by confocal laser scanning microscopy (CLSM), using the fluorescent Ca2+ indicator fluo-3 AM, "cusp"-like structures become visible that are favorably approximated by double parabolae. The time-dependent position of the vertices is used to determine propagation velocity and negative curvature of the wavefront in the region of collision. It is evident that negatively curved waves propagate faster than positively curved, single waves. Considering two perfectly equal expanding circular waves, we demonstrated that the collision of calcium waves is due to an autocatalytic process (calcium-induced calcium release), and not to a simple phenomenon of interference. Following the spatiotemporal organization in simpler chemical systems maintained under conditions far from the thermodynamic equilibrium (Belousov-Zhabotinskii reaction), the dependence of the normal velocity on the curvature of the spreading wavefront is given by a linear relation. The so-called velocity-curvature relationship makes clear that the velocity is enhanced by curvature toward the direction of forward propagation and decreased by curvature away from the direction of forward propagation (with an influence of the diffusion coefficient). Experimentally obtained velocity data of both negatively and positively curved calcium waves were approximated by orthogonal weighted regression. The negative slope of the straight line resulted in an effective diffusion coefficient of 1.2 x 10(-4) mm2/s. From the so-called critical radius, which must be exceeded to initiate a traveling calcium wave, a critical volume (with enhanced [Ca2+]i) of approximately 12 microm3 was calculated. This is almost identical to the volume that is occupied by a single calcium spark.

Effect of ultrasound on the contraction of isolated myocardial cells of adult rats.

Enzymatically isolated myocardial cells of Wistar rats were used to study contraction under the influence of ultrasound. The dynamics of sarcomere length were measured using a laser diffraction technique. The presence of continuous-wave ultrasound (f = 2.25 MHz, PSPTA = 0.3 MPa and f = 10 MHz, PSPTA = 0.15 MPa) did not cause any significant change in sarcomere dynamics (n = 21). However, it was shown that stimulation threshold could be decreased when ultrasound at 10 MHz was applied (14 of 22 cells). Low-frequency ultrasound was not able to cause these alterations. This effect was also found to be dependent on the distance between the sound transducer and the cells. Since temperature effects were negligible and cavitation was very unlikely under the given experimental conditions, we propose that acoustic streaming is responsible for the shift of stimulation threshold.

Developmental changes of calcium transients and contractility during the cultivation of rat neonatal cardiomyocytes.

Neonatal cardiomyocytes of the rat were investigated (a) by Confocal Laser Scanning Microscopy (CLSM) using the Ca(2+)-sensitive dye fluo-3/AM to measure calcium transients, and (b) by a Laser Doppler Microscope (LSC-1) to obtain data of the cell culture's contractility. Our experiments resulted in: (1) About 20% of the freshly prepared cardiomyocytes exhibited spontaneous but not rhythmically appearing calcium transients. None of these cells was found to be active mechanically. The remainder of 80% showed neither calcium transients nor cell movements. (2) At the latest after four days of cultivation, the cells showed spontaneous calcium transients of constant frequency and concomitant contractions. (3) During the cultivation, spontaneous Ca2+ transients became steeper and shorter. The time course of the calcium transient is abbreviated by a factor of at least two in cells after four days when compared with cardiac cells after one day of cultivation. (4) Addition of 100 nM ryanodine caused an increase of the cytosolic calcium concentration and a decrease of the amplitude of the Ca2+ transients. This effect became more significant with increasing time of cultivation and ran parallel to a decrease of the cell's contractility. (5) Addition of 1 microM thapsigargin yielded a similar increase of the cytosolic calcium concentration and a decrease of the Ca2+ peak accompanied by a smaller lowering of the contractility (in comparison with the mentioned influence of ryanodine). The effects of thapsigargin were practically independent of the time of cultivation.

Nonlinear propagation of spherical calcium waves in rat cardiac myocytes.

Spontaneous calcium waves in enzymatically isolated rat cardiac myocytes were investigated by confocal laser scanning microscopy (CLSM) using the fluorescent Ca2+-indicator fluo-3 AM. As recently shown, a spreading wave of enhanced cytosolic calcium appears, most probably during Ca2+ overload, and is initiated by an elementary event called a "calcium spark." When measured by conventional fluorescence microscopy the propagation velocity of spontaneous calcium waves determined at several points along the cardiac myocyte was previously found to be constant. More precise measurements with a CLSM showed a nonlinear propagation. The wave velocity was low, close to the focus, and increased with increasing time and propagation length, approaching a maximum of 113 microns/s. This result was surprising, inasmuch as for geometrical reasons a decrease of the propagation velocity might be expected if the confocal plane is not identical with that plane where the focus of the wave was localized. It is suggested that the propagation velocity is essentially dependent on the curvature of the spreading wave. From the linear relationship of velocity versus curvature, a critical radius of 2.7 +/- 1.4 microns (mean +/- SD) was worked out, below which an outward propagation of the wave will not take place. Once released from a sufficiently extended cluster of sarcoplasmic reticulum release channels, calcium diffuses and will activate its neighbors. While traveling away, the volume into which calcium diffuses becomes effectively smaller than at low radii. This effect is the consequence of the summation of elementary events (Ca2+ sparks) and leads to a steeper increase of the cytosolic calcium concentration after a certain diffusion path length. Thus the time taken to reach a critical threshold of [Ca2+]i at the neighboring calcium release sites decreases with decreasing curvature and the wave will propagate faster.

Shortening-induced tension enhancement: implication for length-tension relations.

Length-tension relations come in two types--the classical type with linearly descending limb, and the "flat," higher type. The classical type, now confirmed in several laboratories, is obtained when sarcomeres are servo-controlled to maintain constant length. The flat type, also confirmed in several laboratories, is obtained in fixed-end contractions, where some sarcomeres have the opportunity to shorten at least slightly. We find that the higher tensions seen in the flat type are indeed the result of very small shortening: when isometric sarcomeres shorten to a slightly shorter (e.g., by 40 nm) length, they go on to produce considerably more isometric tension than if they had remained at the shorter length throughout contraction. We term this phenomenon "shortening-induced tension enhancement." The phenomenon accounts not only for the higher, flatter length-tension relation seen in fixed-end contractions but can explain the creep of tension that occurs in extended tetani. Thus, several issues concerning the interpretation of length-tension relations are resolved by this newly discovered phenomenon.

The effects of thyroid state on sarcomere dynamics of ventricular cells and contraction of papillary muscles in the rat heart.

Diastolic sarcomere length, amplitude of maximal sarcomere shortening, maximal rate of sarcomere shortening, maximal rate of sarcomere re-lengthening, time to peak sarcomere shortening and trans-sarcolemmal ion currents were measured in isolated ventricular cells from euthyroid, hypothyroid and hyperthyroid rats. The data were compared with the developed tension and time to peak tension of papillary muscles. The diastolic sarcomere length was not affected by the changes in thyroid state. Hypothyroidism led to an increased time to peak sarcomere shortening, an increased time to peak tension of the papillary muscle, and a depression of the maximal rates of shortening and elongation of the sarcomeres. Changes in dynamics of the sarcomere and contraction of papillary muscle did not occur in parallel under the influence of hyperthyroidism. In comparison with the euthyroid state, the time to peak tension was shortened and amplitude of shortening of the sarcomere was increased. The time to peak shortening of the sarcomere and developed tension of papillary muscle remained unaltered. In cardiac cells, hypothyroidism was associated with a decreased slow Ca2+ current and hyperthyroidism with an increased slow Ca2+ current. In contrast to euthyroid and hypothyroid cardiac cells, the hyperthyroid cardiomyocytes exhibited a trans-sarcolemmal transient inward current after repolarization. Both hypothyroidism and hyperthyroidism resulted in a depressed potentiation of sarcomere shortening and myocardial developed tension after resting.(ABSTRACT TRUNCATED AT 250 WORDS)

Sarcomere dynamics in isolated cardiac myocytes investigated by high-time resolution laser light diffractometry.

Unattached single cells of the guinea pig myocardium were illuminated with laser light. From the position of the mean of the first order diffraction line at a CCD sensor the sarcomere length was calculated with a time resolution of 1 ms. The maximum velocity of the sarcomere shortening was 2.15 +/- 0.87 microns/s (mean +/- S.E.). Nearly 20% of the cells investigated were found to shorten in a stepwise manner. The intensity profiles of these myocytes deviated from the Gaussian shape at the same time as "pauses" became visible. Therefore, this phenomenon might be due to Bragg effects which result from different sarcomere populations within stepwise shortening cardiac cells.

A study of dynamic properties in isolated myocardial cells by the laser diffraction method.

Laser diffraction patterns were investigated from enzymatically isolated, unattached myocardial cells of guinea-pigs and mice. Experiments were performed at 2.5 mM Ca2+ and room temperature. The mean sarcomere length of resting guinea-pig and mouse myocardial cells amounted to about 1.83 micron and 1.75 micron, respectively. When paced with alternating intervals by field stimulation carefully selected ventricular cells showed transient phenomena. (1) The staircase following a rested state contraction was positive in the case of guinea-pig and negative in the case of mouse myocardial cells; (2) The rested state as compared to the steady state sarcomere shortening of guinea-pig and mouse cardiac myocytes amounted to 35% and 600%, respectively; (3) The interval strength curve of guinea-pig myocardial cells passed through a maximum which was 0.26 +/- 0.06 micron (mean +/- S.D.) at a pacing interval of 2 s whereas myocardial cells of mice showed a rise of shortening with increasing intervals reaching a maximum at the rested state (0.24 +/- 0.08 micron). Results were similar to those obtained from multicellular preparations. We conclude therefore, dynamic properties of multicellular preparations are nicely reflected at the sarcomere level.

Sarcomere dynamics by the laser diffraction method of isolated myocardial cells from guinea pigs.

The sarcomere dynamics of enzymatically isolated cardiac myocytes obtained from guinea pig ventricles was studied by the laser diffraction method (He-Ne laser, 3 mW). Unattached cells were stimulated electrically with a frequency of 0.5 s-1 thus producing stable contraction relaxation cycles for more than five minutes. The extent of shortening varied from cell to cell between 0.05 micron and nearly 0.25 micron/src (src = sarcomere) in the steady state. Starting with a rested state contraction repetitive stimulation resulted in a positive staircase and a nearly threefold extent of shortening after reaching the steady state. Synchronous contractions often consisted of two components that were changed markedly during application of ultrasound (5 W cm-2, 1 MHz). The late component disappeared completely while the first one increased continuously. Differences in extent and velocity of sarcomere shortening are probably due to different contributions of the sarcoplasmic reticulum to the total amount of calcium that controls the myofilament activation.

Simulation by two calcium store models of myocardial dynamic properties: potentiation, staircase, and biphasic tension development.

Most considerations and models concerning myocardial dynamic properties e.g. potentiation and staircase, are based upon the existence of storage structures in the heart muscle cell. The phenomenon of biphasic tension development (or two-component contraction) in heart muscle preparations of several mammalian species suggests that the sarcoplasmic reticulum is one, but by no means the major, source of activator calcium for the contractile system. The simulation of dynamic properties including biphasic tension development was performed in two steps by a simple "two-Ca store-model" and by an "expanded two-Ca store-model" with following results: Increasing potentiation indicated a decrease in the degree of coupling between the Ca stores. A shift of the interval strength curve to lower intervals as well as a decrease of the steady state contraction height implies a decrease of both, the coupling and the leakage time constant. There was no standard relation between staircase phenomena and structure parameters. Analog displays showed a late (or second) component at prolongated stimulation intervals, in the transient phase after a rest period, in the case of perfectly coupled or uncoupled stores, and at great time constant tau p (which characterizes the calcium pump activity). It is concluded that the late component of biphasic tension development is due to direct activation by the transsarcolemmal Ca flux of the myofilaments, whereas the early component is caused by the release of stored calcium. In the absence of an early component neither potentiation nor marked treppe may be expected.

Laser diffraction and speckling studies in skeletal and heart muscle.

The optical setup including a low power helium-neon laser as well as microcomputer system based U 880 for on line investigation of laser diffraction and scattering in skeletal and heart muscle is described. Frog muscle single fibres yield excellent laser diffraction patterns which are best adapted to measurements of the sarcomere dynamics. In the presence of 6 mM caffeine the diffraction disappears gradually and a moving speckle pattern occurs. Diffraction and speckle phenomena are compared of skeletal and heart muscle preparations. From autocorrelation and Fourier transformation of intensity fluctuations ("speckling") in resting mammalian cardiac muscle tissue results a favoured frequency of 1 Hz. It is supposed that clusters of sarcomeres move rhythmically but independent from each other as long as the preparation is not stimulated repetitively.

Effects of iodoacetic acid on relaxation parameters of the rabbit papillary muscle.

Measurements have been made of isometric contractions and diastolic oscillations (after-contractions) in right ventricular rabbit papillary muscles at a temperature of 14.5 degrees C. The relaxation courses can be described as damped harmonic oscillations superposed on an exponential descending carrier function by using a model with six parameters. The influence of iodoacetic acid (10(-4) M) was investigated on contraction and relaxation parameters which result from approximation of the measured force values. The following changes were observed: 1. Decrease of the contraction height as well as of the amplitudes of diastolic oscillations. 2. Increase in the damping ratio and in the period of diastolic oscillations both calculated from relaxation parameters. 3. Diminution of the force range which is passed by the carrier function within 6 s of relaxation time accompanied by a transition to contracture. A fast and a slow Ca sequestration process are supposed to exist. The results indicate that both of them are influenced by iodoacetic acid.

[Effect of the pacing interval and extrasystole on amplitude and damping of diastolic oscillations in the rabbit papillary muscle]. / Einfluss von Treibintervall und Extrasystole auf Amplitude und Dämpfung diastolischer Oszillationen am Kaninchenpapillarmuskel.

Diastolic mechanical oscillations of right ventricular rabbit papillary muscles investigated at 15 degrees C in the perfusion chamber were analysed using a relaxation model with 6 parameters. From this analysis follows that the first diastolic oscillation amplitude plotted against the driving interval ("interval amplitude curve" of the first diastolic oscillation) shows tow maxima thus differing from the shape of the interval strength curve of the preceding driven contraction. It is concluded that the amplitude of diastolic oscillations is less determined by the Ca-amount released from the sarcoplasmic reticulum during the action potential than by the frequency of processes which effect the Ca release and the Ca-sequestration. The damping ratio varies within the range of tested driving intervals (0.36 to 10 s). After an extrasystole damping ratio and period of diastolic oscillation are diminished compared with the values after regularly driven contractions.

What information contains a rest contraction curve? a theoretical study with experimental results from the rabbit papillary muscle.

On the basis of a specialized two-Ca store model the degree of coupling q between the two stores was introduced. q is defined by the time constants of the model (leakage and coupling time constant) and varies between O (uncoupled stores) and 1 (maximal coupled stores). Provided that the contraction height in first approximation is proportional to the myoplasmic Ca concentration from the rest contraction curve the degree of coupling can be calculated using the coordinates of the maximum developed tension. From 13 rabbit papillary muscles the values of q were determined at different periods of rest thus demonstrating that q cannot be considered as constant in the first period of the pause. Accordingly, the time constants change rapidly at the beginning of the rest reaching relatively constant values after the half of the maximum time of the rest contraction curve. With respect to the hypothesis of intracellular recycling Ca controlling the contraction in cardiac muscle the great values of q (about 0.9 after a longer period of rest) suggest a very little loss of Ca from intracellular stores by leakages. From our calculations it is expected that in the maximum of the rest contraction curve the Ca concentration in both stores might differ insignificantly.

[Modelling of relaxation phenomena in the myocardium]. / Modellierung von Relaxationsphänomenen am Herzmuskel.

A model describing relaxation phenomena on heart muscle preparations is proposed, permitting characterization of diastolic oscillations (aftercontractions) that are considered as being damped harmonic oscillations superposed an exponentially descending carrier function (oscillating case). If there is a high damping ratio diastolic oscillations cannot be observed except the phenomenon of hyperrelaxation. Relaxation without oscillations corresponds to the aperiodic case. Examples of relaxation time courses resulting from right ventricular rabbit papillary muscles (isometric conditions) as well as relaxation parameters and approximation curves obtained by the aid of the table computer EMG 666 are given. The model is realized by an electrical analog which is based on a hypothesis concerning the calcium sequestration in the myoplasm.