Journal: bioRxiv

Article Title: Differential contribution of HCN1 and HCN4 to the synchronisation of sinoatrial pacemaker cells

doi: 10.64898/2025.12.16.694405

Figure Lengend Snippet: Averaged phase response curves and AP firing rates differ in isolated SAN cells from WT and HCN1 -/- mice. (A) Spontaneous SAN action potential with brief depolarising current pulses (+50 pA, 20 ms). In HCN1 -/- cells the voltage responses (ΔV) were more pronounced as compared to WT. (B-C) Firing rate and MDP of WT (black) and HCN1 -/- (green) cells showing type 0 and type 1 phase response behaviour during measurements without cAMP in the intracellular solution. (D) Mean type 0 (left panel) and type 1 (right panel) phase response curves of WT (black) and HCN1 -/- (green) SAN cells. (E) Percentage of all cells showing the typical type 0 or type 1 phase response. Boxplots show the median line, perc 25/75, and min/max value; open symbols represent the mean value. Significance levels: Student’s two-sided t-test (* p < 0.05).

Article Snippet: After permeabilization (0.5% Triton X100, 20% DMSO in PBS) and blocking in 5% NDS (Normal Donkey Serum), the tissue was incubated with guinea-pig polyclonal HCN1 antibody (1:500; Alomone Labs, Jerusalem, Israel) and rabbit polyclonal HCN4 antibody (1:500; Alomone Labs).

Techniques: Isolation

Journal: bioRxiv

Article Title: Differential contribution of HCN1 and HCN4 to the synchronisation of sinoatrial pacemaker cells

doi: 10.64898/2025.12.16.694405

Figure Lengend Snippet: The SAN network forms the intrinsic leading pacemaker region of the heart driven by If-conducting cells. (A) Preparation of a gelatine-inflated heart indicating the SAN region (dashed line). (B) Fluorescence imaging of an anti-HCN1 (red signal) and anti-HCN4 (green signal) immunostained whole-mount right atrial preparation of a WT heart, revealing the anatomical extension (head, body, tail region) and spatial HCN1/HCN4 channel expression profile of the SAN. (C) Isolated SAN pacemaker cell (elongated cell) during a patch clamp recording. (D) Family of current traces (If) recorded from a WT pacemaker cell of the sinoatrial node using the voltage protocol shown on the top. From a holding potential of −50 mV, a series of voltage pulses ranging from −140 to - 20 mV (duration 5 s, delta V: 15 mV) were applied. (E) Mean activation curve of native If recorded from WT SAN pacemaker cells (n=13). Additional abbreviations: CT, crista terminalis; SVC, superior vena cava.

Article Snippet: After permeabilization (0.5% Triton X100, 20% DMSO in PBS) and blocking in 5% NDS (Normal Donkey Serum), the tissue was incubated with guinea-pig polyclonal HCN1 antibody (1:500; Alomone Labs, Jerusalem, Israel) and rabbit polyclonal HCN4 antibody (1:500; Alomone Labs).

Techniques: Fluorescence, Imaging, Expressing, Isolation, Patch Clamp, Activation Assay

Journal: bioRxiv

Article Title: Differential contribution of HCN1 and HCN4 to the synchronisation of sinoatrial pacemaker cells

doi: 10.64898/2025.12.16.694405

Figure Lengend Snippet: Computer simulations predict increased HR fluctuations in HCN1 -/- mice but not in HCN4FEA mice. (A) Experimentally determined type 0 and type 1 PRCs (upper panels) were convolved with a murine SAN pacemaker potential model to obtain effective PRCs (lower panels). (B) Effective PRCs were used for simulating the synchronisation behaviour of computational models of WT (left) and HCN1 -/- (right) SAN networks based on the theory of coupled phase oscillators. These simulations were used to determine the expected cycle lengths over 5 minutes. (C) Tachograms were generated by plotting all consecutive cycle lengths (CL) against the time, based on simulations of WT vs. HCN1 -/- mice (left) and WT vs. HCN4FEA mice (right). (D) Poincaré plots were generated by plotting each cycle length (CL+1) against the previous one (CL) to visualise beat-to-beat dispersion in cycle length. HR fluctuations are more pronounced in HCN1 -/- than WT mice. No difference was found between WT and HCN4FEA mice.

Article Snippet: After permeabilization (0.5% Triton X100, 20% DMSO in PBS) and blocking in 5% NDS (Normal Donkey Serum), the tissue was incubated with guinea-pig polyclonal HCN1 antibody (1:500; Alomone Labs, Jerusalem, Israel) and rabbit polyclonal HCN4 antibody (1:500; Alomone Labs).

Techniques: Generated, Dispersion