Journal: Frontiers in Pharmacology

Article Title: HCN4 gain-of-function mutation increases intrinsic heart rate and limits maladaptive remodeling under pressure overload

doi: 10.3389/fphar.2026.1840832

Figure Lengend Snippet: Characterization of the HCN4(Y527F) gain-of-function mutation and generation of the HCN4F mouse model. (A) Schematic depicting two subunits of the HCN4 channel tetramer, highlighting the transmembrane domains (yellow), C-Terminus comprising the cyclic nucleotide-binding domain (CNBD) and C-linker region (purple), and the N-terminal HCN domain (green). (B) Crystal structure of the human HCN4 C-terminal region (PDB: Q9Y3Q4 ) showing the arrangement of the A′ and B′ helices within the C-linker and the localization of the substitution of tyrosine 527 by phenylalanine. (C) Representative If current traces from HEK293 cells stably expressing WT (left) or HCN4F channels (right) under measurement conditions without cAMP. (D) Steady-state activation curves determined from measurements as shown in (C) for WT and HCN4F channels with (open circles) and without cAMP (filled circles). (E) Half-maximum activation voltage (V0.5) determined from steady-state activation curves. (F) Activation kinetics comparison between WT and HCN4F. (G) Mean current density of WT and HCN4F with and without cAMP. (H) Targeting strategy for generation of the HCN4F knock-in mouse line. The HCN4 WT locus comprises exons 3–8. Exon 4 depicted in blue carries the Hcn4Y527 F mutation. Data points in D and E represent the mean ± SEM; n numbers are given in parentheses. Box plots show median (line), 25th–75th percentiles (box), minimum–maximum (whiskers), and mean (open circle). P < 0.05 was considered significant (P < 0.05 *, P < 0.01 **, P < 0.001 ***).

Article Snippet: Tissue was incubated with a guinea-pig polyclonal anti-HCN1 antibody (1:500; Alomone Labs) and rabbit polyclonal anti-HCN4 antibody (1:500; Alomone Labs).

Techniques: Mutagenesis, Binding Assay, Stable Transfection, Expressing, Activation Assay, Comparison, Knock-In

Journal: Frontiers in Pharmacology

Article Title: HCN4 gain-of-function mutation increases intrinsic heart rate and limits maladaptive remodeling under pressure overload

doi: 10.3389/fphar.2026.1840832

Figure Lengend Snippet: Validation of HCN4F channel expression and function in sinoatrial node tissue. (A) Representative whole-mount SAN immunofluorescence images of HCN4 (green) and HCN1 (red) staining from WT (top) and HCN4F (bottom). (B) Representative SAN section immunofluorescence images of HCN4 (green) and HCN1 (red) staining in head (top left), body (top, right) and tail region (bottom) of SAN tissue. (C) Quantification of intranodal HCN1/HCN4 area ratio in the head, body, and tail regions of the SAN. HCN channel distribution and expression levels are similar in WT and HCN4F SAN. (D) Representative families of If current traces recorded from isolated SAN cells of WT and HCN4F mice under baseline and cAMP (100 µM) conditions. (E) If steady-state activation curves determined from recordings as shown in (C,F) Half-maximal activation voltage (V0.5) comparison between WT and HCN4F under baseline and cAMP (100 µM) conditions. (G,H) Activation kinetics (τ1 and τ2) derived from double-exponential fits under baseline and cAMP conditions. (I) Mean If steady-state current density determined at −140 mV. Data are presented as mean ± SEM; n numbers are given in parentheses. SVC, superior caval vein; CT, crista terminalis; RA, right atrial appendage. Data points in E and F represent the mean ± SEM; n numbers are given in parentheses. Box plots show median (line), 25th–75th percentiles (box), minimum–maximum (whiskers), and mean (open circle). P < 0.05 was considered significant (P < 0.05 *, P < 0.01 **, P < 0.001 ***).

Article Snippet: Tissue was incubated with a guinea-pig polyclonal anti-HCN1 antibody (1:500; Alomone Labs) and rabbit polyclonal anti-HCN4 antibody (1:500; Alomone Labs).

Techniques: Biomarker Discovery, Expressing, Immunofluorescence, Staining, Isolation, Activation Assay, Comparison, Derivative 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: 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: The Journal of Physiological Sciences : JPS

Article Title: Doxorubicin-induced sinus node dysfunction associated with mitochondria and nuclear impairment in a mouse model

doi: 10.1016/j.jphyss.2025.100047

Figure Lengend Snippet: Doxorubicin-induced cardiomyopathy modelling in mice. A. A work flow of the modelling of doxorubicin (DXB)-induce cardiomyopathy in C57BL/6 N adult male mice. Saline was injected as a vehicle (Veh) control. B. The time course of the body weight in Veh- and DXB-treated mice. While the body weight of Veh mice incrementally gained, that of DXB mice decreased. n = 6 in Veh and 10 in DXB. C. Heart weight-to-tibial length ratio in Veh- and DXB-treated mice. n = 6 in Veh and 5 in DXB. D and E. Picrosirius red-stained sections (D) and averaged fibrosis area (E) of the left ventricular myocardium in Veh- and DXB-treated mice. n = 4/group. Scale bars = 20 μm. F. Representative picrosirius red-stained sections of the SN region in Veh- or DXB-treated mice and enlarged area labelled with HCN4, a pacemaker channel as a SN marker, indicated by rectangles in picrosirius red images. Scale bars = 100 μm. G. Averaged fibrosis area of the SN region in Veh- and DXB-treated mice. n = 4/group. * p < 0.05 vs Veh, determined by unpaired t -test.

Article Snippet: After blocking with 0.1 % bovine serum albumin in PBS, the sections were incubated overnight at 4 °C with rabbit polyclonal anti-HCN4 antibodies (1:100, APC-052, Alomone Labs).

Techniques: Saline, Injection, Control, Staining, Marker, Immunofluorescence

Journal: The Journal of Physiological Sciences : JPS

Article Title: Doxorubicin-induced sinus node dysfunction associated with mitochondria and nuclear impairment in a mouse model

doi: 10.1016/j.jphyss.2025.100047

Figure Lengend Snippet: Chronic effect of doxorubicin (DXB) on expression levels of genes responsible for pacemaking. A and B. Gene expression levels of HCN4 pacemaker channel and Ca 2+ regulators in the sinus node (A) and the right atrial myocardium (B) in vehicle (Veh)- and DXB-treated mice. n = 6 in SN, n = 3 in right atrium, * p < 0.05 vs Veh determined by unpaired t -test.

Article Snippet: After blocking with 0.1 % bovine serum albumin in PBS, the sections were incubated overnight at 4 °C with rabbit polyclonal anti-HCN4 antibodies (1:100, APC-052, Alomone Labs).

Techniques: Expressing, Gene Expression