Raffaella Milanesi

Heteromeric HCN1–HCN4 channels: a comparison with native pacemaker channels from the rabbit sinoatrial node

‘Funny‐’ (f‐) channels of cardiac sino‐atrial node (SAN) cells are key players in the process of pacemaker generation and mediate the modulatory action of autonomic transmitters on heart rate. The molecular components of f‐channels are the hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channels. Of the four HCN isoforms known, two (HCN4 and HCN1) are expressed in the rabbit SAN at significant levels. However, the properties of f‐channels of SAN cells do not conform to specific features of the two isoforms expressed locally. For example, activation kinetics and cAMP sensitivity of native pacemaker channels are intermediate between those reported for HCN1 and HCN4. Here we have explored the possibility that both HCN4 and HCN1 isoforms contribute to the native If in SAN cells by co‐assembling into heteromeric channels. To this end, we used heterologous expression in human embryonic kidney (HEK) 293 cells to investigate the kinetics and cAMP response of the current generated by co‐transfected (HCN4 + HCN1) and concatenated (HCN4‐HCN1 (4–1) tandem or HCN1‐HCN4 (1–4) tandem) rabbit constructs and compared them with those of the native f‐current from rabbit SAN. 4–1 tandem, but not co‐transfected, currents had activation kinetics approaching those of If; however, the activation range of 4–1 tandem channels was more negative than that of the f‐channel and their cAMP sensitivity were poorer (although that of 1–4 tandem channels was normal). Co‐transfection of 4–1 tandem channels with minK‐related protein 1(MiRP1) did not alter their properties. HCN1 and HCN4 may contribute to native f‐channels, but a ‘context’‐dependent mechanism is also likely to modulate the channel properties in native tissues.

Localization of Pacemaker Channels in Lipid Rafts Regulates Channel Kinetics

Lipid rafts are discrete membrane subdomains rich in sphingolipids and cholesterol. In ventricular myocytes a function of caveolae, a type of lipid rafts, is to concentrate in close proximity several proteins of the β-adrenergic transduction pathway. We have investigated the subcellular localization of HCN4 channels expressed in HEK cells and studied the effects of such localization on the properties of pacemaker channels in HEK and rabbit sinoatrial (SAN) cells. We used a discontinuous sucrose gradient and Western blot analysis to detect HCN4 proteins in HEK and in SAN cells, and found that HCN4 proteins localize to low-density membrane fractions together with flotillin (HEK) or caveolin-3 (SAN), structural proteins of caveolae. Lipid raft disruption by cell incubation with methyl-β-cyclodextrin (MβCD) impaired specific HCN4 localization. It also shifted the midpoint of activation of the HCN4 current in HEK cells and of If in SAN cells to the positive direction by 11.9 and 10.4 mV, respectively. These latter effects were not due to elevation of basal cyclic nucleotide levels because the cholesterol-depletion treatment did not alter the current response to cyclic nucleotides. In accordance with an increased If, MβCD-treated SAN cells showed large increases of diastolic depolarization slope (87%) and rate (58%). We also found that the kinetics of HCN4- and native f-channel deactivation were slower after lipid raft disorganization. In conclusion, our work indicates that pacemaker channels localize to lipid rafts and that disruption of lipid rafts causes channels to redistribute within the membrane and modifies their kinetic properties.

Properties of ivabradine-induced block of HCN1 and HCN4 pacemaker channels

Ivabradine is a ‘heart rate‐reducing’ agent able to slow heart rate, without complicating side‐effects. Its action results from a selective and specific block of pacemaker f‐channels of the cardiac sinoatrial node (SAN). Investigation has shown that block by ivabradine requires open f‐channels, is use dependent, and is affected by the direction of current flow. The constitutive elements of native pacemaker channels are the hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels, of which four isoforms (HCN1–4) are known; in rabbit SAN tissue HCN4 is expressed strongly, and HCN1 weakly. In this study we have investigated the blocking action of ivabradine on mouse (m) HCN1 and human (h) HCN4 channels heterologously expressed in HEK 293 cells. Ivabradine blocked both channels in a dose‐dependent way with half‐block concentrations of 0.94 μm for mHCN1 and 2.0 μm for hHCN4. Properties of block changed substantially for the two channels. Block of hHCN4 required open channels, was strengthened by depolarization and was relieved by hyperpolarization. Block of mHCN1 did not occur, nor was it relieved, when channels were in the open state during hyperpolarization; block required channels to be either closed, or in a transitional state between open and closed configurations. The dependence of block upon current flow was limited for hHCN4, and not significant for mHCN1 channels. In summary our results indicate that ivabradine is an ‘open‐channel’ blocker of hHCN4, and a ‘closed‐channel’ blocker of mHCN1 channels. The mode of action of ivabradine on the two channels is discussed by implementing a simplified version of a previously developed model of f‐channel kinetics.

Interaction of the Pacemaker Channel HCN1 with Filamin A

Pacemaker channels are encoded by the HCN gene family and are responsible for a variety of cellular functions including control of spontaneous activity in cardiac myocytes and control of excitability in different types of neurons. Some of these functions require specific membrane localization. Although several voltage-gated channels are known to interact with intracellular proteins exerting auxiliary functions, no cytoplasmic proteins have been found so far to modulate HCN channels. Through the use of a yeast two-hybrid technique, here we showed that filamin A interacts with HCN1, an HCN isoform widely expressed in the brain, but not with HCN2 or HCN4. Filamin A is a cytoplasmic scaffold protein with actin-binding domains whose main function is to link transmembrane proteins to the actin cytoskeleton. Using several HCN1 C-terminal constructs, we identified a filamin A-interacting region of 22 amino acids located downstream from the cyclic nucleotide-binding domain; this region is not conserved in HCN2, HCN3, or HCN4. We also verified by immunoprecipitation from bovine brain that the filamin A-HCN1 interaction is functional in vivo. In filamin A-expressing cells (filamin+), HCN1 (but not HCN4) channels were expressed in hot spots, whereas they were evenly distributed on the membrane of cells lacking filamin A (filamin–) indicating that interaction with filamin A affects membrane localization. Also, in filamin– cells the gating kinetics of HCN1 were strongly accelerated relative to filamin+ cells. The interaction with filamin A may contribute to localizing HCN1 channels to specific neuronal areas and to modulating channel activity.

Recessive Loss-of-Function Mutation in the Pacemaker HCN2 Channel Causing Increased Neuronal Excitability in a Patient with Idiopathic Generalized Epilepsy

The hyperpolarization-activated Ih current, coded for by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, controls synaptic integration and intrinsic excitability in many brain areas. Because of their role in pacemaker function, defective HCN channels are natural candidates for contributing to epileptogenesis. Indeed, Ih is pathologically altered after experimentally induced seizures, and several independent data indicate a link between dysfunctional HCN channels and different forms of epilepsy. However, direct evidence for functional changes of defective HCN channels correlating with the disease in human patients is still elusive. By screening families with epilepsy for mutations in Hcn1 and Hcn2 genes, we found a recessive loss-of-function point mutation in the gene coding for the HCN2 channel in a patient with sporadic idiopathic generalized epilepsy. Of 17 screened members of the same family, the proband was the only one affected and homozygous for the mutation. The mutation (E515K) is located in the C-linker, a region known to affect channel gating. Functional analysis revealed that homomeric mutant, but not heteromeric wild-type/mutant channels, have a strongly inhibited function caused by a large negative shift of activation range and slowed activation kinetics, effectively abolishing the HCN2 contribution to activity. After transfection into acutely isolated newborn rat cortical neurons, homomeric mutant, but not heteromeric wild type/mutant channels, lowered the threshold of action potential firing and strongly increased cell excitability and firing frequency when compared with wild-type channels. This is the first evidence in humans for a single-point, homozygous loss-of-function mutation in HCN2 potentially associated with generalized epilepsy with recessive inheritance.

Embryonic Stem Cell–Derived CD166 + Precursors Develop Into Fully Functional Sinoatrial-Like Cells

Rationale: A cell-based biological pacemaker is based on the differentiation of stem cells and the selection of a population displaying the molecular and functional properties of native sinoatrial node (SAN) cardiomyocytes. So far, such selection has been hampered by the lack of proper markers. CD166 is specifically but transiently expressed in the mouse heart tube and sinus venosus, the prospective SAN. Objective: We have explored the possibility of using CD166 expression for isolating SAN progenitors from differentiating embryonic stem cells. Methods and Results: We found that in embryonic day 10.5 mouse hearts, CD166 and HCN4, markers of the pacemaker tissue, are coexpressed. Sorting embryonic stem cells for CD166 expression at differentiation day 8 selects a population of pacemaker precursors. CD166+ cells express high levels of genes involved in SAN development (Tbx18, Tbx3, Isl-1, Shox2) and function (Cx30.2, HCN4, HCN1, CaV1.3) and low levels of ventricular genes (Cx43, Kv4.2, HCN2, Nkx2.5). In culture, CD166+ cells form an autorhythmic syncytium composed of cells morphologically similar to and with the electrophysiological properties of murine SAN myocytes. Isoproterenol increases (+57%) and acetylcholine decreases (−23%) the beating rate of CD166-selected cells, which express the &bgr;-adrenergic and muscarinic receptors. In cocultures, CD166-selected cells are able to pace neonatal ventricular myocytes at a rate faster than their own. Furthermore, CD166+ cells have lost pluripotency genes and do not form teratomas in vivo. Conclusions: We demonstrated for the first time the isolation of a nonteratogenic population of cardiac precursors able to mature and form a fully functional SAN-like tissue.

A gain-of-function mutation in the cardiac pacemaker HCN4 channel increasing cAMP sensitivity is associated with familial Inappropriate Sinus Tachycardia.

Aims Inappropriate Sinus Tachycardia (IST), a syndrome characterized by abnormally fast sinus rates and multisystem symptoms, is still poorly understood. Because of the relevance of HCN4 channels to pacemaker activity, we used a candidate-gene approach and screened IST patients for the presence of disease-causing HCN4 mutations. Methods and results Forty-eight IST patients, four of whom of known familial history, were enrolled in the study. We initially identified in one of the patients with familial history the R524Q mutation in HCN4. Investigation extended to the family members showed that the mutation co-segregated with IST-related symptoms. The R524Q mutation is located in the C-linker, a region known to couple cAMP binding to channel activation. The functional relevance of the mutation was investigated in heterologous expression systems by patch-clamp experiments. We found that mutant HCN4 channels were more sensitive to cAMP than wild-type channels, in agreement with increased sensitivity to basal and stimulated adrenergic input and with a faster than normal pacemaker rate. The properties of variant channels indicate therefore that R524Q is a gain-of-function mutation. Increased channel contribution to activity was confirmed by evidence that when spontaneously beating rat newborn myocytes were transfected with R524Q mutant HCN4 channels, they exhibited a faster rate than when transfected with wild-type HCN4 channels. Conclusion This is the first report of a gain-of-function HCN4 mutation associated with IST through increased sensitivity to cAMP-dependent activation.

An LQTS6 MiRP1 Mutation Suppresses Pacemaker Current and is Associated with Sinus Bradycardia

Sinus node (SN) dysfunction is observed in some long‐QT syndrome (LQTS) patients, but has not been studied as a function of LQTS genotype. LQTS6 involves mutations in the hERG β‐subunit MiRP1, which also interacts with hyperpolarization‐activated, cyclic nucleotide gated (HCN) channels—the molecular correlate of SN pacemaker current (If). An LQTS registry search identified a 55‐year male with M54T MiRP1 mutation, history of sinus bradycardia (39–56 bpm), and prolonged QTc.

The genetic basis for inherited forms of sinoatrial dysfunction and atrioventricular node dysfunction

The sinoatrial node (SAN) and the atrioventricular node (AVN) are the anatomical and functional regions of the heart which play critical roles in the generation and conduction of the electrical impulse. Their functions are ensured by peculiar structural cytological properties and specific collections of ion channels. Impairment of SAN and AVN activity is generally acquired,but in some cases familial inheritance has been established and therefore a genetic cause is involved. In recent years, combined efforts of clinical practice and experimental basic science studies have identified and characterized several causative gene mutations associated with the nodal syndromes. Channelopathies, i.e., diseases associated with defective ion channels, remain the major cause of genetically determined nodal arrhythmias; however, it is becoming increasingly evident that mutations in other classes of regulatory and structural proteins also have profound pathophysiological roles. In this review, we will present some aspects of the genetic identification of the molecular mechanism underlying both SAN and AVN dysfunctions with a particular focus on mutations of the Na, pacemaker (HCN), and Ca channels. Genetic defects in regulatory proteins and calcium-handling proteins will be also considered. In conclusion, the identification of the genetic defects associated with familial nodal dysfunction is an essential step for implementing an appropriate therapeutic treatment.

A novel de novo HCN1 loss-of-function mutation in genetic generalized epilepsy causing increased neuronal excitability

The causes of genetic epilepsies are unknown in the majority of patients. HCN ion channels have a widespread expression in neurons and increasing evidence demonstrates their functional involvement in human epilepsies. Among the four known isoforms, HCN1 is the most expressed in the neocortex and hippocampus and de novo HCN1 point mutations have been recently associated with early infantile epileptic encephalopathy. So far, HCN1 mutations have not been reported in patients with idiopathic epilepsy. Using a Next Generation Sequencing approach, we identified the de novo heterozygous p.Leu157Val (c.469C > G) novel mutation in HCN1 in an adult male patient affected by genetic generalized epilepsy (GGE), with normal cognitive development. Electrophysiological analysis in heterologous expression model (CHO cells) and in neurons revealed that L157V is a loss-of-function, dominant negative mutation causing reduced HCN1 contribution to net inward current and responsible for an increased neuronal firing rate and excitability, potentially predisposing to epilepsy. These data represent the first evidence that autosomal dominant missense mutations of HCN1 can also be involved in GGE, without the characteristics of epileptic encephalopathy reported previously. It will be important to include HCN1 screening in patients with GGE, in order to extend the knowledge of the genetic causes of idiopathic epilepsies, thus paving the way for the identification of innovative therapeutic strategies.