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.

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.

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.