Paradigm shift: new concepts for HCN4 function in cardiac pacemaking

Figures

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  Fig. 1. HCN4 is expressed in the sinoatrial node. (A) Right, schematic diagram of the cardiac conduction system (green). The primary pacemaker site is the sinoatrial node (SAN). The atrioventricular node (AVN) is the only electrically conductive connection between the atria and the ventricles. The bundle of His (His) splits into a left and right bundle branch (LBB/RBB, left/right bundle branch), which spread out to the left and right Purkinje fiber (PF) network. Abbreviations: LA, left atrium; RA, right atrium; PV, pulmonary veins; VCS, superior vena cava; VCI, inferior vena cava; CS, coronary sinus; RV, right ventricle; LV, left ventricle; VS, ventricular septum. (A) Left, upper panel: distribution of HCN4 (green) in a transverse section of the murine SAN. HCN4 is expressed across the entire SAN region. Abbreviations: CT, crista terminalis; IAS, interatrial septum. Scale bar: 100 µm. Lower panel: schematic illustration of the original image shown in the upper panel. (B) Action potential recordings of isolated SAN cells demonstrating the chronotropic effect at the single cell level. Input from the sympathetic nervous system accelerates SDD and increases the firing rate of pacemaker…

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  Fig. 2. CDR and hysteresis of HCN4 control the firing mode of SAN cells. (A) Action potential recording of an isolated pacemaker cell showing the typical alternation between firing and nonfiring. The mean membrane potential is more depolarized during firing (~ − 55 mV, green line) and more hyperpolarized during nonfiring (~ − 75 mV, red line). At the same time, slow drifts in membrane potential occur. The firing mode is characterized by a slow, progressive hyperpolarization (Δ = − 7 mV) until firing stops (1), leading to an abrupt drop to significantly more hyperpolarized potentials. Conversely, during nonfiring, a slow and progressive depolarization occurs until the threshold for firing is reached (2), and the membrane potential abruptly jumps to substantially more depolarized values. Due to hysteresis of HCN4, the changes in membrane potential have important consequences for the voltage-dependent activation of the channel. (B) Original steady-state activation curves recorded from HCN4 channels heterologously expressed in HEK239 cells without cAMP in the intracellular solution. The long-lasting, mean membrane potentials during firing (− 55 mV) and nonfiring (− 75 mV) are mimicked by the…

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  Fig. 3. Tonic entrainment in the SAN. (A) Scheme of a SAN cell visualizing the signal transduction pathway following stimulation by the ANS. Gs protein-coupled beta-1-adrenergic receptors (green) are activated by norepinephrine (NE) released from sympathetic nerve terminals. Subsequent Gαs signaling stimulates adenylyl cyclases (ACs, gray) to synthetize cAMP, which directly activates HCN4 channels. Conversely, Gi protein-coupled M2 muscarinic receptors (red) are activated by acetylcholine (ACh) released from vagal nerve terminals. Gαi signaling inhibits ACs and thereby reduces the intracellular cAMP level, leading to a decrease in HCN4 activity. Similar effects are evoked by TRIP8bnano, a synthetic peptide that binds to the CNBD of HCN4 and inhibits cAMP-dependent activation of the channel. (B) cAMP-dependent activation of HCN4 reduces the number of nonfiring cells in the SAN, which stabilizes the network rhythm during HR acceleration. (C) Reduction in cAMP-dependent activation of HCN4 increases the number of nonfiring cells in the SAN. Overshooting inhibition leads to bradycardia and SAN dysrhythmia due to destabilization of the network rhythm. (D) The tonic entrainment process takes…

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  Fig. 4. Cardiac phenotype of HCN4FEA mice. (A) Telemetric ECG trace of an HCN4FEA mouse showing severe sinus dysrhythmia. (B) Mean (left), minimum (middle), and maximum (right) heart rate of WT (black) and HCN4FEA mice (green) calculated from 72-h telemetric ECG recordings. (C) Heart rate histograms determined from 72-h recordings. In HCN4FEA mice, the average HR and full HR range is shifted towards lower HR values, demonstrating intrinsic bradycardia. (D) Comet-shaped Poincaré plots display high beat-to-beat dispersion in HCN4FEA mice (green). (E) Tachograms of WT (black) and HCN4FEA mice (green) before and after consecutive injections of propranolol and atropine. (F) Optical imaging measurements of biatrial SAN explants reveal prolonged sinoatrial conduction time (SACT) in HCN4FEA mice. (G) Quantification of SACT determined from optical measurements as shown in panel (F). (H) Combined telemetric blood pressure (upper panel) and ECG recordings (RR intervals, lower panel) used to determine baroreflex sensitivity in vivo. (I) Plot of systolic blood pressure (SBP) and corresponding RR intervals (upper panel) demonstrates a steeper slope of the RR/SBP relationship in HCN4FEA mice (green),…