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The funny current, I f, was first recorded in the heart 40 or more years ago by Dario DiFrancesco and others. Since then, we have learnt that I f plays an important role in pacemaking in the sinus node, the innate pacemaker of the heart, and more recently evidence has accumulated to show that I f may play an important role in action potential conduction through the atrioventricular (AV) node. Evidence has also accumulated to show that regulation of the transcription and translation of the underlying Hcn genes plays an important role in the regulation of sinus node pacemaking and AV node conduction under normal physiological conditions - in athletes, during the circadian rhythm, in pregnancy, and during postnatal development - as well as pathological states - ageing, heart failure, pulmonary hypertension, diabetes and atrial fibrillation. There may be yet more pathological conditions involving changes in the expression of the Hcn genes. Here, we review the role of I f and the underlying HCN channels in physiological and pathological changes of the sinus and AV nodes and we begin to explore the signalling pathways (microRNAs, transcription factors, GIRK4, the autonomic nervous system and inflammation) involved in this regulation. This review is dedicated to Dario DiFrancesco on his retirement.

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondazione Cariplo Caveolae are small-membrane invagination that contribute both to buffering excessive contraction-dependent membrane strain and to initiation of membrane repair. Moreover, they constitute micro-domains where receptors and ion channels are clustered, favouring their functional interaction. Caveolin-3 (Cav3) is the key structural component of muscular caveolae. Mutations in Cav3 gene are associated with alterations of the skeletal muscle architecture leading to some rare forms of hereditary skeletal myopathies and/or cardiomyopathies called caveolinopathies. Notably, skeletal muscle dysfunctions usually precede cardiac dysfunctions, even though the mutated Cav3 is expressed in both cell types. An important difference between skeletal fibers and cardiomyocytes is that in the latter, caveolin-1 (Cav1) participates with Cav3 to form caveolae; skeletal myotubes instead do not express Cav1.The delay or lack of onset of cardiac alterations in caveolinopathies may depend on a preserved micro-domains organization in the heart compared to skeletal muscle, due to Cav1 expression. We decided to focus on a specific mutation T78K found in heterozygous in a patient with Ripple muscle disease and hyperCKemia. We have characterized human cardiomyocytes (CM) differentiated from induced pluripotent stem cells (iPSC) derived from this patient and one healthy control. In particular, we have investigated which caveolin isoforms are expressed at day 30 of differentiation, finding both Cav1 and Cav3, with a significant decrease of Cav3 isoform in T78K-CM. Their different expressions significantly increase T78K membrane resistance (3.27 ± 0.6 GΩ versus 1.64 ± 0.4 GΩ in the CTRL-CM), and consequently membrane excitability. The T78K_CM showed an increase spontaneous beating rate compared to CTRL (1,75± 0,08 Hz and 0,89 ± 0,4 Hz, respectively). Previous laboratory analysis conducted in caveolin-free MEF cells, co-transfected with WT and T78K Cav3 mutation, revealed that the T78K mutant is dominant, inducing the retention of WT Cav3 in the perinuclear areas and causes significant reduction in current density of three ion channels (HCN4, Kv1.5 and Kir2.1) known to interact with caveolins. The dominant decreased in cav3 expression is in line with previous data in skeletal muscle biopsy, however, electrophysiological data would be likely incompatible with life. For this reason, we decided to compare the impact of this mutation in CHO cells that exhibit high levels of Cav1, and in cav-1 expressing MEF line. In these systems, the membrane localization of Cav3 T78K is rescued both in heterozygous and homozygous conditions. In line with caveolin membrane expression, HCN4, Kv1.5 and Kir2.1 density is also rescued to normal levels. These results constitute the first evidence of a possible role of Cav1 in compensating membrane disorganization and disfunction due to Cav3 mutations in the heart, making this organ less susceptible to caveolinopathies.

Background Heart rate follows a diurnal variation, and slow heart rhythms occur primarily at night. Objective The lower heart rate during sleep is assumed to be neural in origin, but here we tested whether a day-night difference in intrinsic pacemaking is involved. Methods In vivo and in vitro electrocardiographic recordings, vagotomy, transgenics, quantitative polymerase chain reaction, Western blotting, immunohistochemistry, patch clamp, reporter bioluminescence recordings, and chromatin immunoprecipitation were used. Results The day-night difference in the average heart rate of mice was independent of fluctuations in average locomotor activity and persisted under pharmacological, surgical, and transgenic interruption of autonomic input to the heart. Spontaneous beating rate of isolated (ie, denervated) sinus node (SN) preparations exhibited a day-night rhythm concomitant with rhythmic messenger RNA expression of ion channels including hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4). In vitro studies demonstrated 24-hour rhythms in the human HCN4 promoter and the corresponding funny current. The day-night heart rate difference in mice was abolished by HCN block, both in vivo and in the isolated SN. Rhythmic expression of canonical circadian clock transcription factors, for example, Brain and muscle ARNT-Like 1 (BMAL1) and Cryptochrome (CRY) was identified in the SN and disruption of the local clock (by cardiomyocyte-specific knockout of Bmal1) abolished the day-night difference in Hcn4 and intrinsic heart rate. Chromatin immunoprecipitation revealed specific BMAL1 binding sites on Hcn4, linking the local clock with intrinsic rate control. Conclusion The circadian variation in heart rate involves SN local clock–dependent Hcn4 rhythmicity. Data reveal a novel regulator of heart rate and mechanistic insight into bradycardia during sleep.

Brugada syndrome (BrS) is a disorder characterized by cardiac conduction system dysfunctions, which increases the risk of sudden cardiac death, without heart structural alterations. The diagnosis is based on the ECG tracing analysis: BrS patients have common anomalies. The typical ECG pattern, however, can remain latent and patients can be asymptomatic: the first symptom often coincides with death. Recently, a new mutation associated with BrS has been identified and characterized by HEK 293 cells. This study aims to analyse, through numerical simulation of the O ‘Hara-Rudy (ORd) model, the mutation effects on the ventricular action potential (AP). Under normal conditions, the simulation results do not show significant alterations in the mutant AP. For this reason, we hypothesized that the pathological BrS phenotype could be triggered by other factors.

Background: Brugada syndrome (BrS) is a cardiac disorder characterized by conduction abnormalities that can lead to sudden death; syncope and cardiac arrest are clinical manifestations which are often associated with an enhancement of the vagal activity. Mutations in the SCN5A gene (Na v 1.5 channel) are the most common cause of the inherited forms of BrS. Objective: To characterize the functional behavior of mutant Na v 1.5 channels expressing a novel heterozygous mutation (S805L) recently identified in an Italian family affected by the BrS. Methods: HEK cells were used as experimental model to express both the wild-type (WT) and the mutated S805L channels (alone, Homo or in combination, Hetero) and the accessory β-subunit (SCN1B). Patch-clamp and western blot experiments were carried out to assess the dysfunctional role of the mutation. Results: When compared to the WT current, the S508L mutation significantly (P&It0.05) decreases the peak current density by about 65% for the Homo condition (WT: -120.2±10.2, n=28); Homo: -40.3±4.2, n=16) and by 35% for the Hetero condition (Hetero: -78.2±8.3, n=27). Densitometric analysis carried out on western blot data further support the conclusion that S805L channels are less abundant in the plasma membrane. We also observed that the S805L mutation positively shifts the V½ values of the voltage dependence of the inactivation of both Homo and Hetero currents (V½: WT -85.5±0.2 mV, n=55; Homo -80.9±0.3 mV, n=22; Hetero -81.9±0.2 mV, n=25; P&It0.05); a positive shift of the V½ of the activation was also observed but only in the Homo condition (V½: WT -33.0±0.4 mV, n=28; Homo -30.0±0.5, n=16, P&It0.05). The kinetics of recovery from inactivation and the amplitude of the late sodium current were also evaluated but they were unaffected by the mutation. Conclusion: When expressed in the Hetero condition, the S805L mutation causes a reduction in the channel expression, however, the positive shift of the inactivation curve suggests an increase in Na channel availability. We thus believe that the precise quantitative balance between these two phenomena and their relation with vagal activity may underlie the clinical manifestation of the disease.

HCN channels are highly expressed and functionally relevant in neurons and increasing evidence demonstrates their involvement in the etiology of human epilepsies. Among HCN isoforms, HCN4 is important in cardiac tissue, where it underlies pacemaker activity. Despite being expressed also in deep structures of the brain, mutations of this channel functionally shown to be associated with epilepsy have not been reported yet. Using Next Generation Sequencing for the screening of patients with idiopathic epilepsy, we identified the p.Arg550Cys (c.1648C>T) heterozygous mutation on HCN4 in two brothers affected by benign myoclonic epilepsy of infancy. Functional characterization in heterologous expression system and in neurons showed that the mutation determines a loss of function of HCN4 contribution to activity and an increase of neuronal discharge, potentially predisposing to epilepsy. Expressed in cardiomyocytes, mutant channels activate at slightly more negative voltages than wild-type (WT), in accordance with borderline bradycardia. While HCN4 variants have been frequently associated with cardiac arrhythmias, these data represent the first experimental evidence that functional alteration of HCN4 can also be involved in human epilepsy through a loss-of-function effect and associated increased neuronal excitability. Since HCN4 appears to be highly expressed in deep brain structures only early during development, our data provide a potential explanation for a link between dysfunctional HCN4 and infantile epilepsy. These findings suggest that it may be useful to include HCN4 screening to extend the knowledge of the genetic causes of infantile epilepsies, potentially paving the way for the identification of innovative therapeutic strategies.

In mammals cardiac rate is determined by the duration of the diastolic depolarization of sinoatrial node (SAN) cells which is mainly determined by the pacemaker I(f) current. f-channels are encoded by four members of the hyperpolarization-activated cyclic nucleotide-gated gene (HCN1-4) family. HCN4 is the most abundant isoform in the SAN, and its relevance to pacemaking has been further supported by the discovery of four loss-of-function mutations in patients with mild or severe forms of cardiac rate disturbances. Due to its selective contribution to pacemaking, the I(f) current is also the pharmacological target of a selective heart rate-reducing agent (ivabradine) currently used in the clinical practice. Albeit to a minor extent, the I(f) current is also present in other spontaneously active myocytes of the cardiac conduction system (atrioventricular node and Purkinje fibres). In working atrial and ventricular myocytes f-channels are expressed at a very low level and do not play any physiological role; however in certain pathological conditions over-expression of HCN proteins may represent an arrhythmogenic mechanism. In this review some of the most recent findings on f/HCN channels contribution to pacemaking are described.

The pacemaker current, mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, contributes to the initiation and regulation of cardiac rhythm. Previous experiments creating HCN-based biological pacemakers in vivo found that an engineered HCN2/HCN1 chimeric channel (HCN212) resulted in significantly faster rates than HCN2, interrupted by 1–5 s pauses. To elucidate the mechanisms underlying the differences in HCN212 and HCN2 in vivo functionality as biological pacemakers, we studied newborn rat ventricular myocytes over-expressing either HCN2 or HCN212 channels. The HCN2- and HCN212-over-expressing myocytes manifest similar voltage dependence, current density and sensitivity to saturating cAMP concentrations, but HCN212 has faster activation/deactivation kinetics. Compared with HCN2, myocytes expressing HCN212 exhibit a faster spontaneous rate and greater incidence of irregular rhythms (i.e. periods of rapid spontaneous rate followed by pauses). To explore these rhythm differences further, we imposed consecutive pacing and found that activation kinetics of the two channels are slower at faster pacing frequencies. As a result, time-dependent HCN current flowing during diastole decreases for both constructs during a train of stimuli at a rapid frequency, with the effect more pronounced for HCN2. In addition, the slower deactivation kinetics of HCN2 contributes to more pronounced instantaneous current at a slower frequency. As a result of the frequency dependence of both instantaneous and time-dependent current, HCN2 exhibits more robust negative feedback than HCN212, contributing to the maintenance of a stable pacing rhythm. These results illustrate the benefit of screening HCN constructs in spontaneously active myocyte cultures and may provide the basis for future optimization of HCN-based biological pacemakers.

Together with the afferent branches of the autonomic nervous system, the sinoatrial node (SAN) forms a functional unit whose function is to fire rhythmic action potentials at a rate optimal for coping with the metabolic needs of the body. Dysfunctional behavior of this complex unit may thus result in SAN rhythm disorders. Among these disorders, there is the inappropriate sinus tachycardia (IST) which occurs when an unjustified fast SAN rate is present. We here present a critical review of the role of pacemaker f/HCN channels in cardiac rhythm generation and modulation and their involvement in IST. Recent evidence demonstrates that a familial form of IST is associated with a gain-of-function mutation in the HCN4 pacemaker channel (R524Q) which confers an increased sensitivity to the second messenger cAMP, a key mediator in sympathetic modulation. This finding is consistent with the general view that hypersympathetic tone is one of the causes of IST and introduces the novel concept of defective funny channel-dependent tachyarrhythmias.

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.

As opposed to a number of other pharmacological agents, ivabradine expresses high selectivity and specificity for its target. Ivabradine exerts a unique action on cardiac pacemaker activity, based on its block of the hyperpolarization-activated, cyclic nucleotide-gated channels that pass the pacemaker current, If. In doing so, it suppresses but does not stop the sinoatrial pacemaker’s rate of firing. In the following pages, we will review the mechanisms of normal pacemaker activity in the heart, discuss ivabradine’s mechanism of action, and then review the advantages of heart rate reduction in clinical settings as well as other potential applications of the drug.

TMYX is a traditional Chinese Medicine drug used to control cardiac rate in arrhythmic patients. The efficacy and safety of this substance is proved by its clinical use, but the molecular mechanism of action is unknown. TMYX (2 mg/ml) reduces spontaneous rate (−21.9±0.8%, n=11) of pacemaker cardiomyocytes due to a selective reduction of the diastolic depolarization slope (−49.3±3.6%, n=11) caused by a leftward shift of the activation curve of If (−7.1±0.6, n=3) and associated current inhibition.

In the adult animal the sinoatrial node (SAN) rhythmically generates a depolarizing wave that propagates to the rest of the heart. However, the SAN is more than a simple clock; it is a clock that adjusts its pace according to the metabolic requirements of the organism. The Hyperpolarization-activated Cyclic Nucleotide-gated channels (HCN1-4) are the structural component of the funny (I(f)) channels; in the SAN the I(f) current is the main driving electrical force of the diastolic depolarization and the HCN4 is the most abundant isoform. The generation of HCN KO and transgenic mouse models has advanced the understanding of the role of these channels in cardiac excitability. The HCN4 KO models that were first developed allowed either global or cardiac-specific constitutive ablation of HCN4 channels, and resulted in embryonic lethality. A further progress was made with the development of three separate inducible HCN4 KO models; in one model KO was induced globally in the entire organism, in a second, ablation occurred only in HCN4-expressing cells, and finally in a third model KO was confined to cardiac cells. Unexpectedly, the three models yielded different results; similarities and differences among these models will be presented and discussed. The functional effects of HCN2 and HCN3 knockout models and transgenic HCN4 mouse models will also be discussed. In conclusion, HCN KO/transgenic models have allowed to evaluate the functional role of the I(f) currents in intact animals as well as in single SAN cells isolated from the same animals. This opportunity is therefore unique since it allows (1) to verify the contribution of specific HCN isoforms to cardiac activity in intact animals, and (2) to compare these results to those obtained in single cell experiments. These combined studies were not possible prior to the development of KO models. Finally, these models represent critical tools to improve our understanding of the molecular basis of some inheritable arrhythmic human pathologies.

Background A potential concern about biological pacemakers is their possible malfunction, which might create ventricular tachycardias (VTs). Objective The purpose of this study was to test our hypothesis that should VTs complicate implantation of HCN-channel-based biological pacemakers, they would be suppressed by inhibitors of the pacemaker current, I f . Methods We created a chimeric channel (HCN212) containing the N- and C-termini of mouse HCN2 and the transmembrane region of mouse HCN1 and implanted it in HEK293 cells. Forty-eight hours later, in whole-cell patch clamp recordings, mean steady state block induced by 3 μM ivabradine (IVB) showed HCN1=HCN212 > HCN2 currents. The HCN212 adenoviral construct was then implanted into the canine left bundle branch in 11 dogs. Complete AV block was created via radiofrequency ablation, and a ventricular demand electronic pacemaker was implanted (VVI 45 bpm). Electrocardiogram, 24-hour Holter monitoring, and pacemaker log record check were performed for 11 days. Results All dogs developed rapid VT (>120 bpm, maximum rate=285 ± 37 bpm) at 0.9 ± 0.3 days after implantation that persisted through 5 ± 1 days. IVB, 1 mg/kg over 5 minutes, was administered during rapid VT, and three dogs received a second dose 24 hours later. While VT terminated with IBV in all instances within 3.4 ± 0.6 minutes, no effect of IVB on sinus rate was noted. Conclusion We conclude that (1) I f -associated tachyarrhythmias—if they occur with HCN-based biological pacemakers—can be controlled with I f -inhibiting drugs such as IVB; (2) in vitro , IVB appears to have a greater steady state inhibiting effect on HCN1 and HCN212 isoforms than on HCN4; and (3) VT originating from the HCN212 injection site is suppressed more readily than sinus rhythm. This suggests a selectivity of IVB at the concentration attained for ectopic over HCN4-based pacemaker function. This might confer a therapeutic benefit.

The 'funny' current, first described in cardiac pacemaker cells almost 30 years ago, is a key player in the generation of pacemaker activity and the autonomic modulation of heart rate. Because of these specific functions, a search for molecules able to interfere selectively with the 'funny' current was undertaken soon after its discovery, with the aim of developing tools for the pharmacological control of heart rate. This search has succeeded in generating a new class of drugs, the heart rate-reducing agents, which act through specific blockade of f-channels; one of these drugs, ivabradine, is presently marketed against stable angina. Because of their many functions in heart and other tissues, pharmacological utilization of "funny" channel properties is an exciting new frontier open to further developments.

Background— Biological pacemakers (BPM) implanted in canine left bundle branch function competitively with electronic pacemakers (EPM). We hypothesized that BPM engineered with the use of mE324A mutant murine HCN2 (mHCN2) genes would improve function over mHCN2 and that BPM/EPM tandems confer advantage over either approach alone. Methods and Results— In cultured neonatal rat myocytes, activation midpoint was −46.9 mV in mE324A versus −66.1 mV in mHCN2 ( P P Xenopus oocytes. In intact dogs in complete atrioventricular block, saline (control), mHCN2, or mE324A virus was injected into left bundle branch, and EPM were implanted (VVI 45 bpm). Twenty-four–hour ECGs were monitored for 14 days. With EPM discontinued, there was no difference in duration of overdrive suppression among groups. However, basal heart rates in controls were less than those in mHCN2, which did not differ from those in E324A (45 versus 57 versus 53 bpm; P P P Conclusions— mE324A induces faster, more positive pacemaker current activation than mHCN2 and stable, catecholamine-sensitive rhythms in situ that compete with EPM comparably but more catecholamine responsively than mHCN2. BPM/EPM tandems function reliably, reduce the number of EPM beats, and confer sympathetic responsiveness to the tandem.