Fig 2: Immunofluorescence staining and analysis under basal conditions (control) and after 5 days of CVB3 expression (CVB3) in pacemaker-like cells. a Immunofluorescence staining of pacemaker-like cells with antibodies against N-Cadherin (membrane marker) and either anti-HCN4 (top panel), anti-Nav1.5 (middle panel) and anti-Cav1.2 (bottom panel). The distributions were visualized via linescan. b Quantitative distribution analysis of N-Cadherin (n = 30), HCN4 (n = 30), Nav1.5 (n = 5) and Cav1.2 (n = 5). The localization of all targets in the membrane and the cytoplasmatic fraction was monitored and put into a ratiometric dependence (***p < 0.001). c Quantitative expression analysis of N-Cadherin (n = 30), HCN4 (n = 30), Nav1.5 (n = 5) and Cav1.2 (n = 5). The whole fluorescence intensity of all targets in the cells was monitored and put into a percental dependence (**p < 0.01)

Fig 3: Generation and characterization of transgenic HCN4tg/wt mice. a The transgene carries a 4.3 kb promoter fragment of the murine cTnI gene (cTnI) fused to the human HCN4 cDNA and the bovine growth hormone gene poly A signal (BGH-pA). b Profiling of the endogenous murine HCN1 (mHCN1), HCN2 (mHCN2), HCN4 (mHCN4), and transgenic human HCN4 (hHCN4) transcripts in the ventricular myocardium of 2-month-old HCN4tg/wt mice measured by quantitative real-time PCR, (qRT-PCR). Wild type = 1.0 (n = 6 animals; ***P < 0.001; unpaired t-test). c Western blot and quantitative analysis of HCN4 protein in the ventricular myocardium of HCN4tg/wt transgenic and wild type mice, normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for control of protein loading. For comparison samples with different protein load (10, 20, 30 µg) are shown (n = 6 animals/groups; *P < 0.05, **P < 0.01, ***P < 0.001; unpaired t-test). d Immunohistochemistry for HCN4 (red) in ventricular myocardium of wild type (left) and transgene (right) mice. Scale bars 20 µm. Representative If currents (e) and current-voltage relationship (f) recorded from ventricular cardiomyocytes isolated from HCN4tg/wt and wild type mice (n = 20 cells from six wild type mice and n = 16 cells from five HCN4tg/wt mice; *P < 0.05, **P < 0.01, ***P < 0.001; unpaired t-test). Data are expressed as mean ± s.e.m. Source data are provided as a Source data file

Fig 4: Antibody staining in two SA-like cell populations. a–h Immunohistochemical identification of SA-like cells with IF10. Cells with IF10 (green barred arrow) are characterized by the co-localization of HCN1, HCN3, HCN4, and NOS. a A cell with IF10 is labeled by antibodies directed against NOS (NOSpoly). A long (>100 µm) dendrite is visible. b–e Triple-staining for HCN1 (blue HCN1a), NOS (green NOSpoly), and HCN4 (red PG2-1A4). Cells with IF10 show strong expression of NOS, weaker expression of HCN4, and expression around the detection threshold of HCN1. f–h Double-staining for NOS (green NOSpoly) and HCN3 (red HCN3 Shigemoto). i–k Immunohistochemical identification of cells with IF11 (blue barred arrow). These cells express HCN2 (red QQA-1A6). Projection of a confocal LSM section series reveals that dendrites of cells with IF11 do not reach into glomeruli. Glomeruli are visible because of the ramification of vilip1-stained PG-like cells (green vilip1) within glomeruli. Color codes of barred arrows as in Table 2. Bars 10 µm (e, h, k), 50 µm (a)

Fig 5: Expression of HCN subunits in the hippocampal region at E14.5. HCN1 (A), HCN2 (D), and HCN4 (G) immunolabeling was more evident in the intermediate zone (iz) than in the ventricular zone (vz). In the hippocampal iz, labeling for HCN2 and HCN4 subunits was higher than for HCN1. B, E, H: DAPI staining in the nucleus. C, F, I: merged images of HCN immunolabeling and DAPI stain. Scale bars = 20 µm.