Speeding Up the Heart? Traditional and New Perspectives on HCN4 Function

Abstract

The sinoatrial node (SAN) is the primary pacemaker of the heart and is responsible for generating the intrinsic heartbeat. Within the SAN, spontaneously active pacemaker cells initiate the electrical activity that causes the contraction of all cardiomyocytes. The firing rate of pacemaker cells depends on the slow diastolic depolarization (SDD) and determines the intrinsic heart rate (HR). To adapt cardiac output to varying physical demands, HR is regulated by the autonomic nervous system (ANS). The sympathetic and parasympathetic branches of the ANS innervate the SAN and regulate the firing rate of pacemaker cells by accelerating or decelerating SDD-a process well-known as the chronotropic effect. Although this process is of fundamental physiological relevance, it is still incompletely understood how it is mediated at the subcellular level. Over the past 20 years, most of the work to resolve the underlying cellular mechanisms has made use of genetically engineered mouse models. In this review, we focus on the findings from these mouse studies regarding the cellular mechanisms involved in the generation and regulation of the heartbeat, with particular focus on the highly debated role of the hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 in mediating the chronotropic effect. By focusing on experimental data obtained in mice and humans, but not in other species, we outline how findings obtained in mice relate to human physiology and pathophysiology and provide specific information on how dysfunction or loss of HCN4 channels leads to human SAN disease.

Keywords: HCN4 channel; autonomic nervous system; chronotropic effect; cyclic nucleotide-gated (HCN) channels; heart rate regulation; hyperpolarization-activated cation channel; pacemaking; sinoatrial node.

FIGURE 1

The sinoatrial node. (A) Dorsolateral view of the sinoatrial node region of a gelatine-filled mouse heart. (B) Schematic illustration of the heart shown in panel (A), depicting the location of the sinoatrial node (SAN) (gray) within the right atrium (RA). (C, left) Magnification of the SAN region. The cranial portion is referred to as the sinus node “head,” the middle portion as the “body,” and the caudal portion as the “tail.” HCN1 channels are only expressed in the head region whereas HCN4 channels are expressed throughout the whole SAN. (C, right) The SAN is innervated by the sympathetic and parasympathetic nervous system (dashed lines). Activity of both ANS branches tightly controls cAMP concentration in SAN cells. Abbreviations: Ao, Aorta; IVC, inferior vena cava; LCCA, left common carotid artery; LCV, left cranial vein; LSA; left subclavian artery; PA, pulmonary arteries; PV, pulmonary veins; RAA, right atrial appendage; RCCA, right common carotid artery; RSA, right subclavian artery; SVC, superior vena cava. Figure is adapted from Hennis et al., 2021.

FIGURE 2

Membrane and calcium clock contribute to the generation of SDD. (A) Schematic representation of a SAN cell containing the nucleus, cytosol, and sarcoplasmic reticulum (SR). (B) Section of the cell with a magnified view of the plasma membrane and SR membrane. Various proteins that contribute to SDD and are localized in the plasma membrane are collectively described as the membrane clock. These include hyperpolarization-activated cyclic-nucleotide gated (HCN) cation channels (pink), T-type Ca²⁺ channels (green), L-type Ca²⁺ channels (blue), Na⁺/Ca²⁺ exchanger proteins (NCX, yellow), rapid and slow delayed rectifier K⁺ channels (gray), and inward rectifier K⁺ channels (gray). The corresponding ionic currents (I) are indicated. Intracellular Ca²⁺ cycling events that contribute to the pacemaker process are summarized as calcium clock. Among them are sarco-/endoplasmic reticulum Ca²⁺ ATPases (SERCA) (light blue) associated with the regulatory protein phospholamban (PLB) and ryanodine receptors (RyR2) (dark green) located in the SR membrane. (C) Functional interaction of the membrane clock and calcium clock is required to ensure regular and rhythmic excitation of the cells. (D) Spontaneous SAN action potentials with the characteristic slow diastolic depolarization phase (SDD). The maximum diastolic potential (MDP) is indicated. (E) Relative contribution of the ionic currents responsible for SDD and spontaneous action potential firing in mouse SAN cells according to the mathematical model published by Kharche et al. (2011). Current amplitudes are normalized to the cell capacitance (in units of pA/pF). (Upper left panel) I_f manifests as a mainly time-independent, but bidirectionally flowing current. The inward (light purple) and outward component (light blue) are indicated. (Lower right panel) Sarcoplasmic reticulum local calcium releases (SR_LCR) (green) that characteristically occur during late SDD have been added to the model data for cytosolic Ca²⁺ transients according to Lakatta et al. (2010). Abbreviations: I_f, hyperpolarization-activated cyclic nucleotide-gated (HCN) current; I_Ca,T, T-type Ca²⁺ current; I_Ca,L1.2, L-type Ca²⁺ channel isoform Ca_v1.2 current; I_Ca,L1.3, L-type Ca²⁺ channel isoform Ca_v1.3 current; I_Na,1.5, Na⁺ channel isoform Na_v1.5 current; I_K1, inward rectifying K⁺ current; I_K, delayed rectifying K⁺ current; I_NCX, Na⁺/Ca²⁺ exchanger current; Ca²⁺_cytosolic, cytosolic Ca²⁺ concentration; LCR, local calcium release. For further details see text. Figure is adapted from Lakatta et al. (2010), Kharche et al. (2011), Cingolani et al. (2018).

FIGURE 3

Heart rate regulation by the autonomic nervous system. (A) SAN cell action potentials. The intrinsic action potential rate is depicted in gray. Activity of the sympathetic nervous system (orange) leads to a steeper SDD, decreases the time to reach the threshold potential (TP) for the next action potential thereby increasing action potential frequency and consequently also heart rate (positive chronotropic effect). Vagal input flattens SDD, lowers the maximum diastolic potential and thereby increases the time to reach the TP. Action potential frequency and hence HR decrease (negative chronotropic effect). (B) Signaling cascades underlying the chronotropic effect. Release of norepinephrine (NE) from sympathetic nerve terminals leads to activation of Gs protein-coupled β-receptors. Subsequent stimulation of adenylyl cyclases (ACs) increases the cytoplasmic concentration of cAMP. cAMP directly facilitates the opening of HCN channels and activates PKA, which in turn phosphorylates various proteins (indicated by green arrows and green circles), thereby increasing their activity. In addition, sympathetic activity increases the intracellular Ca²⁺ concentration, which increases the activity of NCX and CaMKII. This in turn phosphorylates various target proteins (indicated by purple arrows and circles). Taken together, this steepens the SDD, increases the repolarization rate and thereby increases the action potential frequency (see text for further details). In contrast, the release of acetylcholine (ACh) during vagal activity leads to the activation of Gi protein-coupled M-receptors. ACs are inhibited by the Gi protein and thus the opposite effect of sympathetic activation unfolds. In addition, the β/γ-subunit activates GIRK channels that make the maximum diastolic potential more negative. Abbreviations: ACh, acetylcholine; ACs, adenylyl cyclases; ATP, adenosine triphosphate; CaMKII, Ca²⁺/calmodulin-dependent protein kinase II; GIRK, G protein-coupled inwardly rectifying potassium channels; HCN, hyperpolarization-activated cyclic nucleotide-gated cation channel; NE, norepinephrine; PKA, protein kinase A; PLB, phospholamban; RyR2, ryanodine receptor 2; SERCA, sarco-/endoplasmic reticulum Ca²⁺ ATPase; SR, sarcoplasmic reticulum.