Mitochondriale Calcium-Regulation in isolierten Kardiomyozyten unter normalen und pathologischen Bedingungen

The energy (ATP) required for cardiac contraction is largely (> 90 %) obtained from oxidative phosphorylation in mitochondria. Mitochondria are also involved in the regulation of intracellular calcium (Ca). Due to specialized transport proteins, they are able to take up Ca into their matrix and to remove it back to the cytosol. Mitochondrial Ca (mitoCa) can control both cell metabolism and cell death and is a crucial factor in balancing energy supply and demand in the heart. Therefore, the (impaired) mitoCa regulation could be involved in the development of heart diseases. Thus, a better understanding of mitoCa regulation appears to be mandatory from both physiological and pathological perspectives. In the present study, mitoCa regulation was characterized in isolated cardiomyocytes from adult rats. The investigations were performed in a physiological model (cardiomyocytes from healthy WKY rats) and in three pathological models: Cardiomyocytes from spontaneously hypertensive rats (SHR), cardiomyocytes from haploinsufficient Cacna1c+/- rats and in cardiomyocytes treated with the ER/SR stress inducer tunicamycin. First, a protocol for reliable measurements of mitoCa transients (mitoCaT) was established. For this purpose, the isolated myocytes were loaded with a Ca-dependent fluorescent dye (X-Rhod-1) and the cytosolic fluorescence component was eliminated by CoCl2 quenching. Mitochondrial localization of X-Rhod-1 was confirmed using a specific mitochondrial dye, MitoTracker Green. The cardiomyocytes were electrically stimulated and the resulting mitoCaT were measured on the confocal microscope in linescan mode. Furthermore, they were analyzed and compared with cytosolic Ca transients (cytoCaT, measured with Fluo-4). By suppressing the contraction of the cardiomyocytes using cytochalasin D (10 M), the possibility that the measured mitoCaT were movement artifacts was excluded. In the present study, fast oscillating (beat-to-beat) mitoCaT could be measured in atrial myocytes from WKY rats. During basal stimulation (0,5 Hz) they showed an amplitude of approximately  0,15 – 0,25 (F/Frest), a rise time of approximately  40 – 70 ms and a tau of decay of approximately  60 – 100 ms. By increasing the stimulation frequency, there was an increase in diastolic Ca, a decrease in the amplitude and an acceleration of the tau of decay in the mitoCaT. In addition, substantial subcellular differences were found in the mitoCaT of atrial myocytes. The subsarcolemmal mitoCaT showed considerably larger amplitudes than centrally measured mitoCaT. The comparison with the cytoCaT showed that the (subcellular) mitoCaT resembled the behavior of the (subcellular) cytoCaT but exhibited marked differences with regard to their kinetic parameters. The mitoCaT in atrial myocytes from SHR measured at an early stage of hypertensive heart disease (3 months) also did not (yet) show any changes and were similar to the mitoCaT of healthy WKY rats both globally and subcellularly. However, there were already pronounced differences in the expression of important mitoCa-regulating proteins (NCLX and LETM1 expression increased by  50 %) in the atrial myocardium of SHR, indicating the onset of mitochondrial remodeling in the SHR. With the help of the -adrenergic agonist isoprenaline (30 nM), it was shown that the mitoCaT in ventricular myocytes of Cacna1c+/- rats responded to sympathetic stimulation. There was an increase in diastolic Ca (by  20 %) and in the amplitude of the mitoCaT ( 70 %) as well as an acceleration of the tau of decay (from  105 ms to  75 ms). Despite a previously described altered cytoCa regulation, no significant differences in the mitoCaT of ventricular myocytes were found between the two genotypes (WT vs. Cacna1c+/-). The expression of important mitoCa-regulating proteins (MCU, NCLX, LETM1) in the ventricular myocardium also showed no differences between WT and Cacna1c+/- rats. Finally, the influence of an ER/SR stress inducer (tunicamycin) on mitoCa and cytoCa regulation in ventricular myocytes of WKY rats was investigated. Tunicamycin (10 M) led to severe disturbances of mitoCa and cytoCa regulation within 10 – 12 minutes: In both cases, there was a pronounced increase in diastolic Ca, combined with an increased occurrence of arrhythmogenic Ca waves. In addition, (subcellular) Ca alternans was detected. In summary, fast oscillating (beat-to-beat) mitoCaT could be measured in isolated adult cardiomyocytes, which were regulated in a frequency-dependent manner. A profound difference in subcellular mitoCa regulation between subsarcolemmal and central mitochondria was revealed in atrial myocytes. The two pathology models (SHR and Cacna1c+/-) showed mitoCaT similar to those of healthy controls. Isoprenaline led to larger and faster mitoCaT in ventricular myocytes, as known for cytoCaT. Furthermore, tunicamycin, an ER/SR stress inducer, was shown to cause severe disturbances in cytoCa and mitoCa regulation upon acute administration, ultimately leading to the occurrence of Ca alternans and arrhythmias.