Regulation der cytoplasmatischen und nucleären Calcium-Konzentration durch den speichergesteuerten Calcium-Einstrom in adulten Kardiomyozyten

The store-operated calcium (Ca) entry (SOCE) is a mechanism regulated by the filling state of intracellular Ca stores (endo/sarcoplasmic reticulum, ER/SR). Depletion of the stores leads to the activation of Ca-permeable channels located in the plasma membrane (transient receptor potential canonical (TRPC) and Orai channels) with subsequent Ca influx. The activation of these channels is mediated by the Ca sensor STIM1, which is located in the ER/SR membrane. In electrically nonexcitable cells, SOCE is essential for activating Ca-dependent signaling pathways and refilling of intracellular Ca stores. Cardiomyocytes express SOCE proteins, but their relevance and contribution to Ca-dependent signaling pathways remain elusive. Ca is an important second messenger and plays an essential role for many cellular processes in cardiomyocytes, such as contraction or regulation of energy metabolism. Changes in intracellular Ca concentrations can cause cardiac remodeling. Nuclear Ca is essential for gene transcription in the nucleus. Therefore, the increase of nuclear Ca plays an essential role in hypertrophic gene expression and thus contributes to cardiac remodeling. However, the mechanisms of nuclear Ca regulation are not well understood. SOCE is thought to be involved in remodeling and pathogenesis of cardiac hypertrophy. To date, it is not yet known whether SOCE also affects nuclear Ca. In the present work, adult rat cardiomyocytes were isolated and loaded with a Ca-dependent fluorescent dye (Fluo-4) for functional characterization of SOCE and examined at a confocal microscope. The confocal microscope was used in linescan mode, allowing simultaneous recordings of cytoplasmic and nuclear regions. A protocol for measuring and quantifying SOCE was established first. At the beginning of this protocol, electrically stimulated Ca transients (CaTs) were recorded. Subsequently, the SR was depleted with thapsigargin (0.5 µM) and caffeine (20 mM) in a Ca-free solution. During this procedure, L-type Ca channels (LTCC) were blocked with verapamil (5 µM) and the sodium-calcium exchanger (NCX) was blocked with KB-R7943 (10 µM), thus the proteins that may lead to sarcolemmal Ca influx into the cell were blocked. After store depletion, extracellular Ca was increased and a Ca increase into the cells was recorded. This Ca increase was assessed as SOCE. It could be quantified in both, cytoplasm and nucleus and was blocked by the cation channel blocker gadolinium (1 mM). Electrically stimulated CaTs were used to quantify SOCE such that SOCE amplitude was normalized to CaT amplitude. There was a linear correlation between nuclear and cytoplasmic SOCE, thus the increase of cytoplasmic Ca by SOCE also leads to an increase of nuclear Ca by passive diffusion of Ca through the nuclear pores. The impact of channels contributing to SOCE (TRPC and/or Orai) was studied by using different SOCE inhibitors. For this purpose, the Orai channel inhibitors S66 and BTP-2 and the TRPC channel blocker SKF 96365 were used. S66 (1 µM), BTP-2 (3 µM) and SKF 96365 (5 µM) blocked the Ca influx in the cytoplasm by ≈ 25-40 % and in the nucleus by ≈ 30-40 %. The combination of an Orai channel blocker (S66) with the TRPC channel blocker SKF 96365 led to a reduction of SOCE in the cytoplasm and nucleus by ≈ 60 %. Formamide-induced osmotic shock diminished the T-tubular system of ventricular myocytes. In detubulated ventricular myocytes, SOCE amplitudes in the cytoplasm and nucleus were unchanged. In ventricular myocytes from spontaneously hypertensive rats (SHR) at an early stage of cardiac hypertrophy, SOCE amplitudes were similar to those in ventricular myocytes from normotensive control rats (Wistar-Kyoto-rats, WKY). Atrial and ventricular myocytes show functional and structural differences. Therefore, SOCE was also characterized in atrial myocytes. In these cells, a robust SOCE was also determined in both, the cytoplasm and the nucleus. There was a direct linear correlation between the cytoplasmic and nuclear SOCE in atrial myocytes. Atrial SOCE amplitude was also blocked by the use of pharmacological inhibitors (S66, BTP-2, SKF 96365). Cytoplasmic SOCE amplitudes were reduced by ≈ 65-80 % by the mentioned blockers, nuclear SOCE amplitudes were decreased by ≈ 60-75 %. Cytoplasmic and nuclear SOCE amplitudes were about twice as large in atrial myocytes compared to ventricular myocytes. Western blot studies revealed increased expressions of TRPC1, 3, 6 and STIM1 (≈ 40-260%) in the atrial tissue. The results of this work demonstrate that SOCE in atrial and ventricular cardiomyocytes leads to an increase of cytoplasmic and nuclear Ca. Both, TRPC and Orai channels contribute to the cytosolic and nuclear Ca increase and these channels are predominantly localized in the surface membrane. The increase in nuclear Ca suggests a potential contribution of SOCE to Ca-dependent regulation of transcription during cardiac remodeling.