Mechanisms of zebrafish sinoatrial node (SAN) automaticity. Effects of pharmacological block of membrane (40 min of 3 μM ivabradine hydrochloride for intracellular block of “funny” current, I_f, n = 7; 7 min of 3 mM cesium chloride for extracellular block of I_f, n = 6; 15 min of 165 μM nickel(II) chloride for block of T-type Ca²⁺ current, I_Ca,T, n = 7) or Ca²⁺ (15 min of 1 μM ryanodine for block of ryanodine receptors, n = 6; 30 min exposure of 5 μM BAPTA-AM, followed by 30 min to allow for de-esterification of the AM group, for chelation of cytosolic Ca²⁺, n = 7; 15 min of 0.2 μM nifedipine for block of L-type Ca²⁺ current, I_Ca,L, n = 6) “clock” components, as well as matched time controls (60 min, n = 7) on heart rate (A,C,E) and the inter-beat variability of cycle length (measured as the root mean square of its successive differences, RMSSD_CL; B,D,F), in the isolated zebrafish heart. Representative electrocardiogram (ECG) recording of zebrafish cardiac rhythm in control and after combined pharmacological block of membrane and Ca²⁺ “clock” components (3 mM cesium chloride, 165 μM nickel(II) chloride, and 1 μM ryanodine, n = 6) and measurements of the resulting SAN pause and burst duration and control and burst heart rate (G). Data shown as mean ± SEM. Means compared by paired, two-tailed, Student’s t-tests; *p < 0.05.

Structure and channel expression in the zebrafish sinoatrial node (SAN). Schematic of the zebrafish heart illustrating the location of the images in B-H (A). Overview of the zebrafish SAN complex visualised by eGFP fluorescence in myl7-expressing cardiomyocytes (B). Morphology of SAN cells (C) and atrial myocytes (D) in a 5 μm confocal z-projection. Immunofluorescence of hyperpolarisation-activated cyclic nucleotide-gated channel-4 (HCN4, red; E) and ryanodine receptor (RyR, green; F) expression in a 50 μm confocal z-projection through the SAN and proximal atrial myocardium. Cellular distribution of RyR and HCN4 in a single optical plane from approximately the mid-SAN (G). Scale bars (in μm): (B): 200 μm; (C,D): 25 μm; (E,F): 40 μm; (G): 25 μm. LX, left cardiac branch of the vagal nerve; RX, right cardiac branch of the vagal nerves; SAV, sinoatrial valve.

Effect of vagal nerve stimulation on leading pacemaker site of the zebrafish sinoatrial node (SAN). Schematic of the zebrafish isolated heart showing the location of the SAN and the intact left (LX) and right (RX) cardiac branches of the vagal nerve (A). The leading pacemaker site(s) measured in individual SAN before nerve stimulation (B,E), during left (LVNS, C) or right (RVNS, F) vagal nerve stimulation at 15 Hz, and during LVNS and RVNS after application of the nicotinic receptor blocker hexamethonium (D,G), projected onto a representative SAN image, with individual subjects differentiated by colour (n = 8).

Effects of blebbistatin on the zebrafish sinoatrial node (SAN), atrial, and ventricular action potential (AP). Representative AP (A–C) and measured parameters (D–K) in the SAN (green), atrium (red), and ventricle (blue) with (dark) and without (light) blebbistatin (10 μM for 30 min). APD₅₀ and APD₈₀, AP duration at 50% and 80% repolarisation; DD, diastolic depolarisation; dV/dt_max, maximum rate of change of membrane potential during the AP upstroke; MDP, maximum diastolic potential; V_m, membrane potential. Data shown as mean ± SEM. SAN, atrium, and ventricle compared by unpaired, one-way ANOVA and Tukey post hoc tests; ^#p < 0.05. Control and blebbistatin compared by unpaired, two-tailed, Student’s t-tests; *p < 0.05. n indicates number of impalements, which were acquired in N = 6 hearts.

Effect of mechanical preload on zebrafish and rabbit sinoatrial node automaticity. Representative electrocardiogram (ECG) recording of zebrafish (A) and rabbit (D) sinoatrial node rhythm under unloaded (slack) or mechanically preloaded (5 mg or 300 mg) conditions. Heart rate over time of individual zebrafish (B, n = 8) and rabbit (E, n = 8) sinoatrial nodes when mechanically unloaded or preloaded. Average heart rate and the inter-beat variability of cycle length (measured as the root mean square of its successive differences, RMSSD_CL) of the zebrafish (C) and rabbit (F) sinoatrial node when mechanically unloaded or preloaded. Data shown as mean ± SEM. Means compared by paired, two-tailed, Student’s t-tests; *p < 0.05.

Figure 6. Summary of the drivers of zebrafish sinoatrial node (SAN) automaticity. AP, action potential; DD, diastolic depolarisation; I_Ca,L, L-type Ca²⁺ current; I_Ca,T, T-type Ca²⁺ current; IBI, inter-beat interval; IBV, inter-beat variation; I_f, “funny” current; I_Kr/s, rapid/slow delayed rectifier K⁺ current; I_NCX, Na⁺-Ca²⁺ exchanger current; I_SAC,NS, cation non-selective stretch-activated channels; MDP, maximum diastolic potential; MSP, maximum systolic potential; RyR, ryanodine receptors; SR, sarcoplasmic reticulum; ZF, zebrafish.