Structural determinants of ivabradine block of the open pore of HCN4.

HCN1-4 channels are the molecular determinants of the If/Ih current that crucially regulates cardiac and neuronal cell excitability. HCN dysfunctions lead to sinoatrial block (HCN4), epilepsy (HCN1), and chronic pain (HCN2), widespread medical conditions awaiting subtype-specific treatments. Here, we address the problem by solving the cryo-EM structure of HCN4 in complex with ivabradine, to date the only HCN-specific drug on the market. Our data show ivabradine bound inside the open pore at 3 Å resolution. The structure unambiguously proves that Y507 and I511 on S6 are the molecular determinants of ivabradine binding to the inner cavity, while F510, pointing outside the pore, indirectly contributes to the block by controlling Y507. Cysteine 479, unique to the HCN selectivity filter (SF), accelerates the kinetics of block. Molecular dynamics simulations further reveal that ivabradine blocks the permeating ion inside the SF by electrostatic repulsion, a mechanism previously proposed for quaternary ammonium ions.

A high affinity switch for cAMP in the HCN pacemaker channels.

Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramolecular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity.

Alkali metal cations modulate the geometry of different binding sites in HCN4 selectivity filter for permeation or block.

Hyperpolarization-activated cyclic-nucleotide gated (HCN) channels are important for timing biological processes like heartbeat and neuronal firing. Their weak cation selectivity is determined by a filter domain with only two binding sites for K+ and one for Na+. The latter acts as a weak blocker, which is released in combination with a dynamic widening of the filter by K+ ions, giving rise to a mixed K+/Na+ current. Here, we apply molecular dynamics simulations to systematically investigate the interactions of five alkali metal cations with the filter of the open HCN4 pore. Simulations recapitulate experimental data like a low Li+ permeability, considerable Rb+ conductance, a block by Cs+ as well as a punch through of Cs+ ions at high negative voltages. Differential binding of the cation species in specific filter sites is associated with structural adaptations of filter residues. This gives rise to ion coordination by a cation-characteristic number of oxygen atoms from the filter backbone and solvent. This ion/protein interplay prevents Li+, but not Na+, from entry into and further passage through the filter. The site equivalent to S3 in K+ channels emerges as a preferential binding and presumably blocking site for Cs+. Collectively, the data suggest that the weak cation selectivity of HCN channels and their block by Cs+ are determined by restrained cation-generated rearrangements of flexible filter residues.

Silvio Weidmann: laying the foundations for unravelling the mechanism of heart rhythm.

Silvio Weidmann laid the basis of cardiac electrophysiology and was the forerunner in the search for mechanisms governing the electrical activity of the heart in his legendary first studies of Purkinje fibres in the 1950s. His work was the cornerstone of research in this field for many generations, and countless cardiologists and electrophysiologists have based their studies on the knowledge generated by Weidmann's pioneering data. This review summarizes his key contributions from the first intracellular recordings of cardiac membrane potentials in 1949 to the publication of his monograph in 1956. That summary is followed by an imagined dialogue between the authors and Silvio Weidmann himself, in the format of a one-act play. Both of us have such good recollections of our real-life conversations with Silvio Weidmann that we decided we could achieve a better feel for the history and issues by using a dialogue format. We hope that, in that way, we may transmit the character of Silvio Weidmann better for those readers who will not have known him personally. Silvio Weidmann was an extraordinarily sensitive and conversational person as well as a great scientist, and we feel it is worth the effort to convey that fact here. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Chinese natural compound decreases pacemaking of rabbit cardiac sinoatrial cells by targeting second messenger regulation of f-channels.

Tongmai Yangxin (TMYX) is a complex compound of the Traditional Chinese Medicine (TCM) used to treat several cardiac rhythm disorders; however, no information regarding its mechanism of action is available. In this study we provide a detailed characterization of the effects of TMYX on the electrical activity of pacemaker cells and unravel its mechanism of action. Single-cell electrophysiology revealed that TMYX elicits a reversible and dose-dependent (2/6 mg/ml) slowing of spontaneous action potentials rate (-20.8/-50.2%) by a selective reduction of the diastolic phase (-50.1/-76.0%). This action is mediated by a negative shift of the If activation curve (-6.7/-11.9 mV) and is caused by a reduction of the cyclic adenosine monophosphate (cAMP)-induced stimulation of pacemaker channels. We provide evidence that TMYX acts by directly antagonizing the cAMP-induced allosteric modulation of the pacemaker channels. Noticeably, this mechanism functionally resembles the pharmacological actions of muscarinic stimulation or ß-blockers, but it does not require generalized changes in cytoplasmic cAMP levels thus ensuring a selective action on rate. In agreement with a competitive inhibition mechanism, TMYX exerts its maximal antagonistic action at submaximal cAMP concentrations and then progressively becomes less effective thus ensuring a full contribution of If to pacemaker rate during high metabolic demand and sympathetic stimulation.

Discovery of Herbacetin as a Novel SGK1 Inhibitor to Alleviate Myocardial Hypertrophy.

Cardiac hypertrophy is a pivotal pathophysiological step of various cardiovascular diseases, which eventually leads to heart failure and death. Extracts of Rhodiola species (Ext.R), a class of commonly used medicinal herbs in Europe and East Asia, can attenuate cardiac hypertrophy both in vitro and in vivo. Serum/glucocorticoid regulated kinase 1 (SGK1) is identified as a potential target of Ext. R. By mass spectrometry-based kinase inhibitory assay, herbacetin (HBT) from Ext.R is identified as a novel SGK1 inhibitor with IC50 of 752 nmol. Thermal shift assay, KINOMEscan in vitro assay combined with molecular docking proves a direct binding between HBT and SGK1. Site-specific mutation of Asp177 in SGK1 completely ablates the inhibitory activity of HBT. The presence of OH groups at the C-3, C-8, C-4' positions of flavonoids is suggested to be favorable for the inhibition of SGK1 activity. Finally, HBT significantly suppresses cardiomyocyte hypertrophy in vitro and in vivo, reduces reactive oxygen species (ROS) synthesis and calcium accumulation. HBT decreases phosphorylation of SGK1 and regulates its downstream forkhead box protein O1 (FoxO1) signaling pathway. Taken together, the findings suggest that a panel of flavonoids structurally related to HBT may be novel leads for developing new therapeutics against cardiac hypertrophy.

Age-Related Changes in Cardiac Autonomic Modulation and Heart Rate Variability in Mice.

OBJECTIVE: The aim of this study was to assess age-related changes in cardiac autonomic modulation and heart rate variability (HRV) and their association with spontaneous and pharmacologically induced vulnerability to cardiac arrhythmias, to verify the translational relevance of mouse models for further in-depth evaluation of the link between autonomic changes and increased arrhythmic risk with advancing age. METHODS: Heart rate (HR) and time- and frequency-domain indexes of HRV were calculated from Electrocardiogram (ECG) recordings in two groups of conscious mice of different ages (4 and 19 months old) (i) during daily undisturbed conditions, (ii) following peripheral ß-adrenergic (atenolol), muscarinic (methylscopolamine), and ß-adrenergic + muscarinic blockades, and (iii) following ß-adrenergic (isoprenaline) stimulation. Vulnerability to arrhythmias was evaluated during daily undisturbed conditions and following ß-adrenergic stimulation. RESULTS: HRV analysis and HR responses to autonomic blockades revealed that 19-month-old mice had a lower vagal modulation of cardiac function compared with 4-month-old mice. This age-related autonomic effect was not reflected in changes in HR, since intrinsic HR was lower in 19-month-old compared with 4-month-old mice. Both time- and frequency-domain HRV indexes were reduced following muscarinic, but not ß-adrenergic blockade in younger mice, and to a lesser extent in older mice, suggesting that HRV is largely modulated by vagal tone in mice. Finally, 19-month-old mice showed a larger vulnerability to both spontaneous and isoprenaline-induced arrhythmias. CONCLUSION: The present study combines HRV analysis and selective pharmacological autonomic blockades to document an age-related impairment in cardiac vagal modulation in mice which is consistent with the human condition. Given their short life span, mice could be further exploited as an aged model for studying the trajectory of vagal decline with advancing age using HRV measures, and the mechanisms underlying its association with proarrhythmic remodeling of the senescent heart.

Gating movements and ion permeation in HCN4 pacemaker channels.

The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current If/Ih that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg2+ coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K+/Na+ permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.

A circadian clock in the sinus node mediates day-night rhythms in Hcn4 and heart rate.

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.

Human iPSC modelling of a familial form of atrial fibrillation reveals a gain of function of If and ICaL in patient-derived cardiomyocytes.

AIMS: Atrial fibrillation (AF) is the most common type of cardiac arrhythmias, whose incidence is likely to increase with the aging of the population. It is considered a progressive condition, frequently observed as a complication of other cardiovascular disorders. However, recent genetic studies revealed the presence of several mutations and variants linked to AF, findings that define AF as a multifactorial disease. Due to the complex genetics and paucity of models, molecular mechanisms underlying the initiation of AF are still poorly understood. Here we investigate the pathophysiological mechanisms of a familial form of AF, with particular attention to the identification of putative triggering cellular mechanisms, using patient's derived cardiomyocytes (CMs) differentiated from induced pluripotent stem cells (iPSCs). METHODS AND RESULTS: Here we report the clinical case of three siblings with untreatable persistent AF whose whole-exome sequence analysis revealed several mutated genes. To understand the pathophysiology of this multifactorial form of AF we generated three iPSC clones from two of these patients and differentiated these cells towards the cardiac lineage. Electrophysiological characterization of patient-derived CMs (AF-CMs) revealed that they have higher beating rates compared to control (CTRL)-CMs. The analysis showed an increased contribution of the If and ICaL currents. No differences were observed in the repolarizing current IKr and in the sarcoplasmic reticulum calcium handling. Paced AF-CMs presented significantly prolonged action potentials and, under stressful conditions, generated both delayed after-depolarizations of bigger amplitude and more ectopic beats than CTRL cells. CONCLUSIONS: Our results demonstrate that the common genetic background of the patients induces functional alterations of If and ICaL currents leading to a cardiac substrate more prone to develop arrhythmias under demanding conditions. To our knowledge this is the first report that, using patient-derived CMs differentiated from iPSC, suggests a plausible cellular mechanism underlying this complex familial form of AF.

HCN ion channels and accessory proteins in epilepsy: genetic analysis of a large cohort of patients and review of the literature.

The Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are highly expressed in the Central Nervous Systems, where they are responsible for the Ih current. Together with specific accessory proteins, these channels finely regulate neuronal excitability and discharge activity. In the last few years, a substantial body of evidence has been gathered showing that modifications of Ih can play an important role in the pathogenesis of epilepsy. However, the extent to which HCN dysfunction is spread among the epileptic population is still unknown. The aim of this work is to evaluate the impact of genetic mutations potentially affecting the HCN channels' activity, using a NGS approach. We screened a large cohort of patients with epilepsy of unknown etiology for mutations in HCN1, HCN2 and HCN4 and in genes coding for accessory proteins (MiRP1, Filamin A, Caveolin-3, TRIP8b, Tamalin, S-SCAM and Mint2). We confirmed the presence of specific mutations of HCN genes affecting channel function and predisposing to the development of the disease. We also found several previously unreported additional genetic variants, whose contribution to the phenotype remains to be clarified. According to these results and data from literature, alteration of HCN1 channel function seems to play a major role in epilepsy, but also dysfunctional HCN2 and HCN4 channels can predispose to the development of the disease. Our findings suggest that inclusion of the genetic screening of HCN channels in diagnostic procedures of epileptic patients should be recommended. This would help pave the way for a better understanding of the role played by Ih dysfunction in the pathogenesis of epilepsy.

A Brief History of Pacemaking.

Cardiac pacemaking is a most fundamental cardiac function, thoroughly investigated for decades with a multiscale approach at organ, tissue, cell and molecular levels, to clarify the basic mechanisms underlying generation and control of cardiac rhythm. Understanding the processes involved in pacemaker activity is of paramount importance for a basic physiological knowledge, but also as a way to reveal details of pathological dysfunctions useful in the perspective of a therapeutic approach. Among the mechanisms involved in pacemaking, the "funny" (If) current has properties most specifically fitting the requirements for generation and control of repetitive activity, and has consequently received the most attention in studies of the pacemaker function. Present knowledge of the basic mechanisms of pacemaking and the properties of funny channels has led to important developments of clinical relevance. These include: (1) the successful development of heart rate-reducing agents, such as ivabradine, able to control cardiac rhythm and useful in the treatment of diseases such as coronary artery disease, heart failure and tachyarrhythmias; (2) the understanding of the genetic basis of disorders of cardiac rhythm caused by HCN channelopathies; (3) the design of strategies to implement biological pacemakers based on transfer of HCN channels or of stem cell-derived pacemaker cells expressing If, with the ultimate goal to replace electronic devices. In this review, I will give a brief historical account of the discovery of the funny current and the development of the concept of If-based pacemaking, in the context of a wider, more complex model of cardiac rhythmic function.

A Loss-of-Function HCN4 Mutation Associated With Familial Benign Myoclonic Epilepsy in Infancy Causes Increased Neuronal Excitability.

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.

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.

A synthetic peptide that prevents cAMP regulation in mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.

Binding of TRIP8b to the cyclic nucleotide binding domain (CNBD) of mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels prevents their regulation by cAMP. Since TRIP8b is expressed exclusively in the brain, we envisage that it can be used for orthogonal control of HCN channels beyond the central nervous system. To this end, we have identified by rational design a 40-aa long peptide (TRIP8bnano) that recapitulates affinity and gating effects of TRIP8b in HCN isoforms (hHCN1, mHCN2, rbHCN4) and in the cardiac current If in rabbit and mouse sinoatrial node cardiomyocytes. Guided by an NMR-derived structural model that identifies the key molecular interactions between TRIP8bnano and the HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8bnano) which successfully prevented ß-adrenergic activation of mouse If leaving the stimulation of the L-type calcium current (ICaL) unaffected. TRIP8bnano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers.

Generation of human induced pluripotent stem cells (EURACi001-A, EURACi002-A, EURACi003-A) from peripheral blood mononuclear cells of three patients carrying mutations in the CAV3 gene.

Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3), encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs) from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies. Resource table.

Preferential myofibroblast differentiation of cardiac mesenchymal progenitor cells in the presence of atrial fibrillation.

Atrial fibrillation (AF) is characterized by electrical, contractile, and structural remodeling mediated by interstitial fibrosis. It has been shown that human cardiac mesenchymal progenitor cells (CMPCs) can be differentiated into endothelial, smooth muscle, and fibroblast cells. Here, we have investigated, for the first time, the contribution of CMPCs in the fibrotic process occurring in AF. As expected, right auricolae samples displayed significantly higher fibrosis in AF vs control (CTR) patients. In tissue samples of AF patients only, double staining for c-kit and the myofibroblast marker α-smooth muscle actin (α-SMA) was detected. The number of c-kit-positive CMPC was higher in atrial subepicardial regions of CTR than AF cells. AF-derived CMPC (AF-CMPC) and CTR-derived CMPC (Ctr-CMPC) were phenotypically similar, except for CD90 and c-kit, which were significantly more present in AF and CTR cells, respectively. Moreover, AF showed a lower rate of population doubling and fold enrichment vs Ctr-CMPC. When exogenously challenged with the profibrotic transforming growth factor-ß1 (TGF-ß1), AF-CMPC showed a significantly higher nuclear translocation of SMAD2 than Ctr-CMPC. In addition, TGF-ß1 treatment induced the upregulation of COL1A1 and COL1A2 in AF-CMPC only. Further, both a marked production of soluble collagen and α-SMA upregulation have been observed in AF-CMPC only. Finally, electrophysiological studies showed that the inwardly rectifying potassium current (IK1) was evenly present in AF- and Ctr-CMPC in basal conditions and similarly disappeared after TGF-ß1 exposure. All together, these data suggest that AF steers the resident atrial CMPC compartment toward an electrically inert profibrotic phenotype.

The expression of the rare caveolin-3 variant T78M alters cardiac ion channels function and membrane excitability.

AIMS: Caveolinopathies are a family of genetic disorders arising from alterations of the caveolin-3 (cav-3) gene. The T78M cav-3 variant has been associated with both skeletal and cardiac muscle pathologies but its functional contribution, especially to cardiac diseases, is still controversial. Here, we evaluated the effect of the T78M cav-3 variant on cardiac ion channel function and membrane excitability. METHODS AND RESULTS: We transfected either the wild type (WT) or T78M cav-3 in caveolin-1 knock-out mouse embryonic fibroblasts and found by immunofluorescence and electron microscopy that both are expressed at the plasma membrane and form caveolae. Two ion channels known to interact and co-immunoprecipitate with the cav-3, hKv1.5 and hHCN4, interact also with T78M cav-3 and reside in lipid rafts. Electrophysiological analysis showed that the T78M cav-3 causes hKv1.5 channels to activate and inactivate at more hyperpolarized potentials and the hHCN4 channels to activate at more depolarized potentials, in a dominant way. In spontaneously beating neonatal cardiomyocytes, the expression of the T78M cav-3 significantly increased action potential peak-to-peak variability without altering neither the mean rate nor the maximum diastolic potential. We also found that in a small cohort of patients with supraventricular arrhythmias, the T78M cav-3 variant is more frequent than in the general population. Finally, in silico analysis of both sinoatrial and atrial cell models confirmed that the T78M-dependent changes are compatible with a pro-arrhythmic effect. CONCLUSION: This study demonstrates that the T78M cav-3 induces complex modifications in ion channel function that ultimately alter membrane excitability. The presence of the T78M cav-3 can thus generate a susceptible substrate that, in concert with other structural alterations and/or genetic mutations, may become arrhythmogenic.