Manfred Wussling

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 titins MIR, followed by reactivity along the titin molecule in the course of the disease.

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 Ca2+-sensitive dye fluo-3/AM to measure calcium transients, and (b) by a Laser Doppler Microscope (LSC-1) to obtain data of the cell cultures 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 cells contractility. (5) Addition of 1 μM 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.

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 Ca2+-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 receptors 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.

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.

The velocity of calcium waves is expected to depend non-monotoneously on the density of the calcium release units.

In this paper we develop a reaction-diffusion system describing the calcium dynamics in an agarose gel system with resuspended vesicles from the sarcoplasmic reticulum (SR vesicles). We focus on a simple model: compared with living cells (e.g. cardiac myocytes) an important property of the agarose gel system is the absence of the sarcolemma and the spatial separation of the calcium release units (CRUs). Our model includes the kinetics of ryanodine sensitive receptors (RyRs), the activity of the SERCA pumps and the diffusion of free calcium. We describe numerical simulations which show a biphasic relationship between the density of the CRUs and the propagation velocity of spreading waves. The non-monotony can be explained by changes in the amplitude of the local calcium concentration. We formulate implications for the in vitro system which could be verified in future experiments.

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.