Data Availability StatementAll datasets generated because of this study are included in the article/supplementary material. Noble, 2012). As early as 1983, Irishawa and Morad showed in elegant experiments that full inhibition of If current by caesium did not significantly influence SAN spontaneous activity arguing for mechanisms other than If (Noma et al., 1983). On the other hand, other studies suggest a fundamental role of the exchanger in normal automaticity. A low-sodium bath solution inhibited spontaneous action potentials (AP) firing in guinea-pig SAN cells suppressing normal function Tmem1 of NCX (Sanders et al., 2006). Other studies reported that depletion of SR store by application of ryanodine markedly disturbed the normal pacemaker activity in rabbit SAN cells (Bogdanov et al., 2001). Mouse genetic models revealed that partial atrial NCX1 knock out (90%) caused severe bradycardia and other rhythm disorders (Herrmann et al., 2013), while complete atrial NCX knock-out completely suppressed the atrial depolarization exerting ventricular escape rhythm on the ECG (Groenke et al., 2013). The application of KB-R7943, a non-selective NCX inhibitor, also suppressed spontaneous beating in guinea-pig SAN cells (Sanders et al., LY2140023 cost 2006) however it has also marked effect on the Ca2+-currents. The supposed crucial role of NCX in the normal pacemaker function of SAN could not be directly investigated experimentally so far due to the lack of a selective NCX inhibitor. Recently, two novel NCX inhibitors were synthesized: ORM-10103 and ORM-10962, both showing improved selectivity without influencing ICaL function (Jost et al., 2013; Kohajda et al., 2016; Oravecz et al., 2017). In this study we confirmed the contributing role of NCX to spontaneous pacemaking by its direct pharmacological inhibition the novel, selective inhibitor ORM-10962. Our data suggest that a strong crosstalk between If and NCX also exists in multicellular level, which was described and discussed by the Lakatta group earlier in single cell level (Yaniv et al., 2015). In addition, however, extending these earlier findings, we show that the effect of individual If and NCX inhibition is minimal whereas a LY2140023 cost combined inhibition acts synergistically, providing an important safety margin for secure spontaneous activity of the SAN. Materials and Methods Ethical Statement All experiments were conducted in compliance with the (USA NIH publication No 85-23, revised 1996) and conformed to Directive 2010/63/EU of the European Parliament. The protocols had been accepted by the Review Panel of the Section of Animal Health insurance and Meals Control of the Ministry of Agriculture and Rural Advancement, Hungary (XIII./1211/2012). Pets The measurements had been performed in best atrial tissue extracted from youthful New-Zealand white rabbits from both genders weighing 2.0C2.5 kg. Voltage-Clamp Measurements Cell Arrangements For calculating If pacemaker current, we isolated one cells through the SAN area of rabbit center by enzymatic dissociation. The pets had been sacrificed by concussion after getting 400 IU/kg heparin intravenously. The upper body was opened as well as the center was quickly taken out and positioned into cool (4C) option with the next structure (mM): NaCl 135, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidity (HEPES) 10, NaHCO3 4.4, blood sugar 10, CaCl2 1.8, (pH 7.2 with NaOH). The center was mounted on the customized, 60 cm high Langendorff column and perfused with oxygenated and prewarmed (37C) option mentioned previously. After cleaning out of bloodstream (3C5 min) the center was perfused with LY2140023 cost nominally Ca-free option until the center stopped defeating (approx. 3C4 min). The digestive function was performed by perfusion using the same option supplemented with 1.8 mg/ml (260 U/ml) collagenase (type II, Worthington). After 10C12 min, the center was taken off the cannula. The proper atrium was cut as well as the crista terminalis and SAN region were cut and excised into small strips. Strips were positioned into enzyme free of charge option formulated with 1 mM CaCl2 and equilibrated at 37C for 10 min. After 10 min with soft agitation, the cells had been separated by filtering through a nylon mesh. Sedimentation was useful for harvesting cells. The supernatant was taken out.