Ivabradine hydrochloride

To analyze the influence of pharmacological substances on the spontaneous beating rate, we treated cardiac pacemaker cells differentiated by protocol E with 1 µM isoproterenol hydrochloride (Sigma-Aldrich) (n = 6 wells of a six-well plate), 1 µM carbachol (Sigma-Aldrich) (n = 6 wells of a six-well plate) and 3 µM ivabradine hydrochloride (Sigma-Aldrich) (n = 6 wells of a six-well plate).

The HCN inhibitors ZD7288 (Z3777) and ivabradine hydrochloride (SML 0281) were purchased from Sigma-Aldrich. ZD7288 and ivabradine were dissolved in distilled water, and stock solutions of 100 mM or 50 mM were stored at −20°C. Cesium chloride was dissolved in distilled water, and 1 M of the solution was stored at room temperature. The stock solution was diluted with ND66 solution to achieve the final concentration on the day of the experiment.
For zebrafish, the HCN inhibitors at a final concentration of 0.01–1 mM were applied to egg water in which the fish were kept, starting at 24 h post-fertilization (hpf). Heart rates were recorded before (0 h) and at 24 h (24 h) and 48 h (48 h) after the inhibitors had been applied.

Ivabradine hydrochloride, ZD7288 hydrate, NA, histamine, pyrilamine maleate, CsCl, and BSA were purchased from Sigma‐Aldrich. Stock solutions were made in deionized water and stored in aliquots at −20°C until use. PSS had the following composition (in mM): NaCl 119; CaCl₂ 1.6; KCl 4.7; MgSO₄ 1.17; NaHCO₃ 25; KH₂PO₄ 1.18; EDTA 0.026; glucose 5.5. In 125 mM K⁺ solution (KPSS) all NaCl is substituted with KCl, while the 50 mM K⁺ solution was made by mixing appropriate volumes of PSS and KPSS. For immunofluorescence, sterile solutions of phosphate‐buffered saline (PBS) (in mM) 138 NaCl, 2.67 KCl, 8.1 Na₂HPO₄, 1.47 KH₂PO₄, and TEG‐buffer (pH 9) composed of 10 mM tris base and 5 mM EGTA were used.