Pacemaker Current Research Articles

Mama and papa of the early spontaneous electrical activity of the embryo sinoatrial node

In adult mammalian hearts the spontaneous diastolic depolarization of the sinoatrial node (SAN) cells is generated by two interacting systems, a so‐called “membrane‐clock” and “calcium‐clock”. Hyperpolarization‐activated pacemaker current (If) plays a key role in the “membrane‐clock”, while the “calcium‐clock” is based on rhythmic spontaneous Ca2+ release from sarcoplasmic reticulum via ryanodine receptors (RyRs). These complementing systems provide regular pacemaking even in case of damage of one of them. The aim of this study was to evaluate a role of If current and ryanodine receptors in the electrical activity of the developing mouse SAN.MethodsWe used embryos of mice, aged of E9.5, E12.5 and newborn to perform our experiments. After release from the amniotic membrane, the embryos were transferred with a pipette to a clean Petri dish with oxygenated physiological solution containing (in mM/L) 140 NaCl, 5.4 KCl, 0.1 CaCl2, 0.33 NaH2PO4, 1 MgCl2, 10 HEPES, 10 Glucose, (pH 7.38) at room temperature. To record spontaneous action potential (AP) we used conventional microelectodes technique at 37◦C. Differences were considered significant at p < 0.05ResultsMicroelectrode mapping at E9.5 revealed that the SAN cells are located in the inflow tract of the venous pole. At E12 the SAN cells are located in the cavo‐auricular junction. In neonatal mice, SAN cells were detected at intercaval area near to the crista terminalis. At E9.5, in 6 experiments, on 25‐ 30 minute exposure of 3 mkM ivabradine (Iva) an irregularity in AP generation by SAN cells was occurred. The same effects were obtained in 4 experiments on 20‐30 minutes exposure of 3 mkM ryanodine. At E12.5 exposure of Iva 3 mkM for 30 minutes reduced the pacemaker firing rate by 34% (p < 0.05) but did not break the rhythmic generation. In contrast, the exposure of 3 mkM ryanodine for 30 minutes, in 4 experiments, still caused an irregularity in AP generation by SAN cells. In neonatal isolated SAN preparations, exposure of Iva 3 mkM and 3 mkM ryanodine for 30 minutes reduced the pacemaker firing rate equally (on 32%; p < 0.05). However, co‐application of Iva and ryanodine induced an irregularity in AP generation by SAN cells (Figure).Numerical experiments within the framework of mouse SANC model (Kharche, AJP, 11) adopted for embryonic cells (Markov, IEEE, 21) have shown the decrease in the frequency of the AP generation with decreasing of the “funny” currents permeability caused by Iva action.The supposed complete RyRs opening, caused by a relatively low ryanodine concentration, led to a decrease of the AP generation frequency, but not to the rhythm disturbances. However a decrease in RyRs permeability by an order of magnitude led to the appearance of “bursts‐like” irregularity in AP generation.ConclusionThus, the membrane‐clock and the Ca2+‐clock mechanisms is critical for spontaneous electrical activity E9.5 mouse embryos, whereas the Ca2+‐clock mechanisms is critical for increase rate AP generation in development SAN cells.

Ion behavior in the selectivity filter of HCN1 channels

Hyperpolarization-activated cyclic-nucleotide gated channels (HCNs) are responsible for the generation of pacemaker currents (If or Ih) in cardiac and neuronal cells. Despite the overall structural similarity to voltage-gated potassium (Kv) channels, HCNs show much lower selectivity for K+ over Na+ ions. This increased permeability to Na+ is critical to their role in membrane depolarization. HCNs can also select between Na+ and Li+ ions. Here, we investigate the unique ion selectivity properties of HCNs using molecular-dynamics simulations. Our simulations suggest that the HCN1 pore is flexible and dilated compared with Kv channels with only one stable ion binding site within the selectivity filter. We also observe that ion coordination and hydration differ within the HCN1 selectivity filter compared with those in Kv and cyclic-nucleotide gated channels. Additionally, the C358T mutation further stabilizes the symmetry of the binding site and provides a more fit space for ion coordination, particularly for Li+.

Weak Cation Selectivity in HCN Channels Results From K+-Mediated Release of Na+ From Selectivity Filter Binding Sites.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate the pacemaker current which plays an important role in the timing of various biological processes like the heart beat. We used umbrella sampling to explore the potential of mean force for the conduction of potassium and sodium through the open HCN4 pore. Our data explain distinct functional features like low unitary conductance and weak selectivity as a result of high energetic barriers inside the selectivity filter of this channel. They exceed the 3-5 kJ/mol threshold which is presumed as maximal barrier for diffusion-limited conductance. Furthermore, simulations provide a thermodynamic explanation for the weak cation selectivity of HCN channels that contain only two ion binding sites in the selectivity filter (SF). We find that sodium ions bind more strongly to the SF than potassium and are easier released by binding of potassium than of another sodium. Hence ion transport and selectivity in HCN channels is not determined by the same mechanism as in potassium-selective channels; it rather relies on sodium as a weak blocker that can only be released by potassium.

Altered cyclic nucleotide binding and pore opening in a diseased human HCN4 channel

A growing number of nonsynonymous mutations in the human HCN4 channel gene, the major component of the funny channel of the sinoatrial node, are associated with disease but how they impact channel structure and function, and, thus, how they result in disease, is not clear for any of them. Here, we study the S672R mutation, in the cyclic nucleotide-binding domain of the channel, which has been associated with an inherited bradycardia in an Italian family. This may be the best studied of all known mutations, yet the underlying molecular and atomistic mechanisms remain unclear and controversial. We combine measurements of binding by isothermal titration calorimetry to a naturally occurring tetramer of the HCN4 C-terminal region with a mathematical model to show that weaker binding of cAMP to the mutant channel contributes to a lower level of facilitation of channel opening at submicromolar ligand concentrations but that, in general, facilitation occurs over a range that is similar between the mutant and wild-type because of enhanced opening of the mutant channel when liganded. We also show that the binding affinity for cGMP, which produces the same maximum facilitation of HCN4 opening as cAMP, is weaker in the mutant HCN4 channel but that, for both wild-type and mutant, high-affinity binding of cGMP occurs in a range of concentrations below 1 μM. Thus, binding of cGMP to the HCN4 channel may be relevant normally in vivo and reduced binding of cGMP, as well as cAMP, to the mutant channel may contribute to the reduced resting heart rate observed in the affected family.

Contribution of the funny current to the fight-or-flight heart rate increase

Sinoatrial node myocytes are cardiac pacemaker cells that fire spontaneous action potentials (APs) to set heart rate. Sympathetic stimulation accelerates the AP firing rate to match heart rate to changing metabolic demands. The role of the funny current (If) in the generation of spontaneous sinoatrial APs and in their sympathetic response has been debated for decades. We used the AP-clamp technique to directly measure If during sinoatrial APs in the absence and presence of β-adrenergic receptor- (βAR-) stimulation.

Yohimbine Directly Induces Cardiotoxicity on Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Yohimbine is a highly selective and potent α2-adrenoceptor antagonist, which is usually treated as an adjunction for impotence, as well for weight loss and natural bodybuilding aids. However, it was recently reported that Yohimbine causes myocardial injury and controversial results were reported in the setting of cardiac diseases. Here, we used human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model system to explore electrophysiologic characterization after exposure to Yohimbine. HiPSC-CMs were differentiated by employment of inhibitory Wnt compounds. For analysis of electrophysiological properties, conventional whole-cell patch-clamp recording was used. Specifically, spontaneous action potentials, pacemaker currents (If), sodium (Na+) channel (INa), and calcium (Ca++) channel currents (ICa) were assessed in hiPSC-CMs after exposure to Yohimbine. HiPSC-CMs expressed sarcomeric-α-actinin and MLC2V proteins, as well as exhibited ventricular-like spontaneous action potential waveform. Yohimbine inhibited frequency of hiPSC-CMs spontaneous action potentials and significantly prolonged action potential duration in a dose-dependent manner. In addition, rest potential, threshold potential, amplitude, and maximal diastolic potential were decreased, whereas APD50/APD90 was prolonged. Yohimbine inhibited the amplitude of INa in low doses (IC50 = 14.2μM, n = 5) and inhibited ICa in high doses (IC50 = 139.7μM, n = 5). Whereas Yohimbine did not affect the activation curves, treatment resulted in left shifts in inactivation curves of both Na+ and Ca++ channels. Here, we show that Yohimbine induces direct cardiotoxic effects on spontaneous action potentials of INa and ICa in hiPSC-CMs. Importantly, these effects were not mediated by α2-adrenoceptor signaling. Our results strongly suggest that Yohimbine directly and negatively affects electrophysiological properties of human cardiomyocytes. These findings are highly relevant for potential application of Yohimbine in patients with atrioventricular conduction disorder.

Abstract 14141: Role of Pak1 in Sinus Node Function

Introduction: Sinus node (SN) dysfunction (SND) and atrial arrhythmia frequently occur simultaneously with a hazard ratio of 4.2 for new onset atrial fibrillation (AF) in SND patients. We demonstrated previously that loss of p21-activated kinase 1 (Pak1), a serine/threonine kinase increases the propensity for AF by increasing cellular production of reactive oxygen species (ROS). Since pacemaker current (HCN4) is negatively regulated by cellular ROS we hypothesized that loss of Pak1 leads to attenuated SN function. Methods: Heart rate regulation was quantified in wild type (WT) and Pak1 deficient mice (Pak1 -/- ) in vivo and the isolated Langendorff perfused heart using ECGs, bipolar and multi-electrode array derived atrial electrograms. Unpaired t-test was used to compare means between groups. Results: In vivo WT and Pak1 -/- animals exhibited no difference in basal heart rate (WT: 450 ± 26 bpm, n=16; Pak1 -/- : 440 ± 24 bpm, n=13), but intrinsic HR after autonomic blockage, was reduced in Pak1 -/- (WT: 399 ± 32 bpm, n=16; Pak1 -/- : 365 ± 34 bpm, n=13, p<0.05). The result was confirmed in the Langendorff configuration (WT: 359 ± 47 bpm, n=12; Pak1 -/- : 293 ± 47 bpm, n=21, p<0.01) suggesting attenuated SN impulse generation. To determine the contribution of the Ca clock, hearts were perfused with cyclopiazonic acid (5 μM) a blocker of the sarcoplasmic reticulum Ca ATPase. CPA attenuated the HR in WT and Pak1 -/- (WT: -19.6 ± 6.6 % n=2 ; Pak1 -/- : -15.8 ± 7.5 %, n=5) but did not eliminate the difference in HR (WT: 320 ± 37 bpm, n=2; Pak1 -/- : 255 ± 26 bpm, n=5, p<0.05). Block of HCN4 by ivabradine (3 μM), reduced the HR in WT and Pak1 -/- (WT: -45.1 ± 6.3 %, n=6; Pak1 -/- : -22.2 ± 8.6 %, n=7, p<0.001) and eliminated differences in spontaneous activity (WT: 186 ± 43 bpm, n=6; Pak1 -/- : 234 ± 41 bpm, n=7). In vivo the change in HR in response to ß-blocker propranolol (WT: -13.5 ± 2.5 %, n=6; Pak1 -/- : -18.4 ± 1.5 %; n=4, p<0.01) and muscarinic receptor blocker atropine (WT: 9.7 ± 5.0%, n=10; Pak1 -/- : 4.6 ± 4.4 %; n=9, p<0.05) revealed that the attenuated SN impulse generation in Pak1 -/- mice was compensated by an increased sympathetic and attenuated parasympathetic tone. Conclusion: Attenuated Pak1 expression results in suppressed SN impulse generation likely by ROS dependent attenuation of HCN4 expression.

Abstract 13939: Triiodothyronine and Dexamethasone Enhances Electrophysiological Maturation of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Background: Human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) technology opens new avenues for disease modeling and drug development. However, hiPSC-CMs remain functionally immature, which hinders their utility as a model of human cardiomyocytes. Objective: To improve the electrophysiological maturation of hiPSC-CMs. Methods and Results: Day 16 th hiPSC-CMs were treated with vehicle or 100 nmol/L triiodothyronine (T3) and 1 μmol/L Dexamethasone (Dex) for 14 days. On day 30 th , hiPSC-CMs were dissociated and replated as cell sheets or single cells. Optical mapping and patch-clamp technique were used to examine the electrophysiological properties of vehicle- and T3+Dex-treated hiPSC-CMs. T3+Dex-treated hiPSC-CMs had a slower spontaneous beating rate (vehicle 17 ± 0.8 vs . T3+Dex 12 ± 0.8 beats/min, P < 0.01), more hyperpolarized resting membrane potential (vehicle -65.04 ± 1.20 vs. T3+Dex -72.00 ± 1.10 mV, P < 0.01), faster maximal upstroke velocity (vehicle 86.08 ± 8.12 vs. T3+Dex 131.78 ± 8.49 mV/ms, P < 0.01), and shorter action potential duration at 90% repolarization (APD 90 , vehicle 550.44 ± 39.54 vs. T3+Dex 418.59 ± 21.58 ms, P < 0.01) compared to the vehicle. Changes in spontaneous activity and AP were mediated by decreasing the funny current (I f , vehicle -12.20 ± 2.15 vs. T3+Dex -6.98 ± 0.89 pA/pF at -120 mV, P < 0.05) and increasing the inward rectifier potassium current (I K1 , vehicle -4.62 ± 0.59 pA/pF vs . T3+Dex -18.79 ± 1.98 pA/pF at -120 mV, P < 0.01), the sodium current (I Na , vehicle -9.8 ± 1.86 vs. T3+Dex -44.9 ± 7.10 pA/pF at -30 mV, P < 0.01), and the rapidly and slowly activating delayed rectifier potassium currents (I Kr , 0.81 ± 0.07 vs. T3+Dex: 1.35 ± 0.19 pA/pF at 20 mV, P < 0.01; I Ks , vehicle 0.38 ± 0.06 vs. T3+Dex 0.72 ± 0.17 pA/pF at 40 mV, P < 0.05). Furthermore, T3+Dex-treated hiPSC-CM cell sheets (hiPSC-CCSs) exhibited a faster conduction velocity and shorter APD than the vehicle. Inhibition of I K1 by 100 μM BaCl 2 significantly slowed conduction velocity and prolonged APD in T3+Dex-treated hiPSC-CCSs but had no effect in the vehicle group, demonstrating the important role of I K1 for conduction velocity and APD. Conclusion: T3+Dex treatment is an effective approach to rapidly enhance electrophysiological maturation of hiPSC-CMs.

Molecular Remodeling of Cardiac Sinus Node Associated with Acute Chagas Disease Myocarditis.

Chagas disease principally affects Latin-American people, but it currently has worldwide distribution due to migration. Death among those with Chagas disease can occur suddenly and without warning, even in those who may not have evidence of clinical or structural cardiac disease and who are younger than 60 years old. HCN4 channels, one of the principal elements responsible for pacemaker currents, are associated with cardiac fetal reprogramming and supraventricular and ventricular arrhythmias, but their role in chagasic arrhythmias is not clear. We found that a single-dose administration of ivabradine, which blocks HCN4, caused QTc and QRS enlargement and an increase in P-wave amplitude and was associated with ventricular and supraventricular arrhythmias in mice challenged with isoproterenol, a chronotropic/ionotropic positive agent. Continuous treatment with ivabradine did not alter the QTc interval, but P-wave morphology was deeply modified, generating supraventricular arrhythmias. In addition, we found that repolarization parameters improved with ivabradine treatment. These effects could have been caused by the high HCN4 expression observed in auricular and ventricular tissue in infected mice. Thus, we suggest, for the first time, that molecular remodeling by overexpression of HCN4 channels may be related to supraventricular arrhythmias in acute Chagas disease, causing ivabradine over-response. Thus, ivabradine treatment should be administered with caution, while HCN4 overexpression may be an indicator of heart failure and/or sudden death risk.

Cyclic nucleotide-regulated channels (CNG) in GtoPdb v.2021.3

Cyclic nucleotide-gated (CNG) channels are responsible for signalling in the primary sensory cells of the vertebrate visual and olfactory systems. CNG channels are voltage-independent cation channels formed as tetramers. Each subunit has 6TM, with the pore-forming domain between TM5 and TM6. CNG channels were first found in rod photoreceptors [83, 120], where light signals through rhodopsin and transducin to stimulate phosphodiesterase and reduce intracellular cyclic GMP level. This results in a closure of CNG channels and a reduced ‘dark current’. Similar channels were found in the cilia of olfactory neurons [181] and the pineal gland [71]. The cyclic nucleotides bind to a domain in the C terminus of the subunit protein: other channels directly binding cyclic nucleotides include hyperolarisation-activated, cyclic nucleotide-gated channels (HCN), ether-a-go-go and certain plant potassium channels.The HCN channels are cation channels that are activated by hyperpolarisation at voltages negative to ~-50 mV. The cyclic nucleotides cyclic AMP and cyclic GMP directly bind to the cyclic nucleotide-binding domain of HCN channels and shift their activation curves to more positive voltages, thereby enhancing channel activity. HCN channels underlie pacemaker currents found in many excitable cells including cardiac cells and neurons [64, 192]. In native cells, these currents have a variety of names, such as Ih, Iq and If. The four known HCN channels have six transmembrane domains and form tetramers. It is believed that the channels can form heteromers with each other, as has been shown for HCN1 and HCN4 [2]. High resolution structural studies of CNG and HCN channels has provided insight into the the gating processes of these channels [139, 146, 140]. A standardised nomenclature for CNG and HCN channels has been proposed by the NC-IUPHAR Subcommittee on voltage-gated ion channels [108].

Dexmedetomidine Exerts a Negative Chronotropic Action on Sinoatrial Node Cells Through the Activation of Imidazoline Receptors.

Dexmedetomidine (DEX), an α2-adrenoreceptor (α2-AR) and imidazoline receptor agonist, is most often used for the sedation of patients in the intensive care unit. Its administration is associated with an increased incidence of bradycardia; however, the precise mechanism of DEX-induced bradycardia has yet to be fully elucidated. This study was undertaken to examine whether DEX modifies pacemaker activity and the underlying ionic channel function through α2-AR and imidazoline receptors. The whole-cell patch-clamp techniques were used to record action potentials and related ionic currents of sinoatrial node cells in guinea pigs. DEX (≥10 nM) reduced sinoatrial node automaticity and the diastolic depolarization rate. DEX reduced the amplitude of hyperpolarization-activated cation current (If or Ih) the pacemaker current, even within the physiological pacemaker potential range. DEX slowed the If current activation kinetics and caused a significant shift in the voltage dependence of channel activation to negative potentials. In addition, efaroxan, an α2-AR and imidazoline I1 receptor antagonist, attenuated the inhibitory effects of DEX on sinoatrial node automaticity and If current activity, whereas yohimbine, an α2-AR-selective antagonist, did not. DEX did not affect the current activities of other channels, including rapidly and slowly activating delayed rectifier K+ currents (IKr and IKs), L-type Ca2+ current (ICa,L), Na+/Ca2+ exchange current (INCX), and muscarinic K+ current (IK,ACh). Our results indicate that DEX, at clinically relevant concentrations, induced a negative chronotropic effect on the sinoatrial node function through the downregulation of If current through an imidazoline I1 receptor other than the α2-AR in the clinical setting.

The funny current: Even funnier than 40 years ago. Uncanonical expression and roles of HCN/f channels all over the body

Discovered some 40 years ago, the If current has since been known as the “pacemaker” current due to its role in the initiation and modulation of the heartbeat and of neuronal excitability. But this is not all, the funny current keeps entertaining the researchers; indeed, several data discovering novel and uncanonical roles of f/HCN channel are quickly accumulating. In the present review, we provide an overview of the expression and cellular functions of HCN/f channels in a variety of systems/organs, and particularly in sour taste transduction, hormones secretion, activation of astrocytes and microglia, inhibition of osteoclastogenesis, renal ammonium excretion, and peristalsis in the gastrointestinal and urine systems. We also analyzed the role of HCN channels in sustaining cellular respiration in mitochondria and their participation to mitophagy under specific conditions. The relevance of HCN currents in undifferentiated cells, and specifically in the control of stem cell cycle and in bioelectrical signals driving left/right asymmetry during zygote development, is also considered. Finally, we present novel data concerning the expression of HCN mRNA in human leukocytes.We can thus conclude that the emerging evidence presented in this review clearly points to an increasing interest and importance of the “funny” current that goes beyond its role in cardiac sinoatrial and neuronal excitability regulation.

Kinetic modelling of voltage-dependent gating in funny channels

Kinetic modelling of voltage-dependent gating in funny channels

Regulation of sinoatrial funny channels by cyclic nucleotides: From adrenaline and IK2 to direct binding of ligands to protein subunits

The funny current, and the HCN channels that form it, are affected by the direct binding of cyclic nucleotides. Binding of these second messengers causes a depolarizing shift of the activation curve, which leads to greater availability of current at physiological membrane voltages. This review outlines a brief history on this regulation and provides some evidence that other cyclic nucleotides, especially cGMP, may be important for the regulation of the funny channel in the heart. Current understanding of the molecular mechanism of cyclic nucleotide regulation is also presented, which includes the notions that full and partial agonism occur as a consequence of negatively cooperative binding. Knowledge gaps, including a potential role of cyclic nucleotide-regulation of the funny current under pathophysiological conditions, are included. The work highlighted here is in dedication to Dario DiFrancesco on his retirement.

Bidirectional flow of the funny current (If) during the pacemaking cycle in murine sinoatrial node myocytes

Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current (If) is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. Here, we used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of -30 mV. Despite operating at only 2 to 5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that β-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously expressed HCN4 channels and by mathematical models of If Modeling further suggested that the slow rates of activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions.

Remodeling of the Purkinje Network in Congestive Heart Failure in the Rabbit.

Purkinje fibers (PFs) control timing of ventricular conduction and play a key role in arrhythmogenesis in heart failure (HF) patients. We investigated the effects of HF on PFs. Echocardiography, electrocardiography, micro-computed tomography, quantitative polymerase chain reaction, immunohistochemistry, volume electron microscopy, and sharp microelectrode electrophysiology were used. Congestive HF was induced in rabbits by left ventricular volume- and pressure-overload producing left ventricular hypertrophy, diminished fractional shortening and ejection fraction, and increased left ventricular dimensions. HF baseline QRS and corrected QT interval were prolonged by 17% and 21% (mean±SEMs: 303±6 ms HF, 249±11 ms control; n=8/7; P=0.0002), suggesting PF dysfunction and impaired ventricular repolarization. Micro-computed tomography imaging showed increased free-running left PF network volume and length in HF. mRNA levels for 40 ion channels, Ca2+-handling proteins, connexins, and proinflammatory and fibrosis markers were assessed: 50% and 35% were dysregulated in left and right PFs respectively, whereas only 12.5% and 7.5% changed in left and right ventricular muscle. Funny channels, Ca2+-channels, and K+-channels were significantly reduced in left PFs. Microelectrode recordings from left PFs revealed more negative resting membrane potential, reduced action potential upstroke velocity, prolonged duration (action potential duration at 90% repolarization: 378±24 ms HF, 249±5 ms control; n=23/38; P<0.0001), and arrhythmic events in HF. Similar electrical remodeling was seen at the left PF-ventricular junction. In the failing left ventricle, upstroke velocity and amplitude were increased, but action potential duration at 90% repolarization was unaffected. Severe volume- followed by pressure-overload causes rapidly progressing HF with extensive remodeling of PFs. The PF network is central to both arrhythmogenesis and contractile dysfunction and the pathological remodeling may increase the risk of fatal arrhythmias in HF patients.

Regulation of sinus node pacemaking and atrioventricular node conduction by HCN channels in health and disease

The funny current, If, was first recorded in the heart 40 or more years ago by Dario DiFrancesco and others. Since then, we have learnt that If 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 If 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 If 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.

The funny current in genetically modified mice

Since its first description in 1979, the hyperpolarization-activated funny current (If) has been the object of intensive research aimed at understanding its role in cardiac pacemaker activity and its modulation by the sympathetic and parasympathetic branches of the autonomic nervous system. If was described in isolated tissue strips of the rabbit sinoatrial node using the double-electrode voltage-clamp technique. Since then, the rabbit has been the principal animal model for studying pacemaker activity and If for more than 20 years. In 2001, the first study describing the electrophysiological properties of mouse sinoatrial pacemaker myocytes and those of If was published. It was soon followed by the description of murine myocytes of the atrioventricular node and the Purkinje fibres. The sinoatrial node of genetically modified mice has become a very popular model for studying the mechanisms of cardiac pacemaker activity. This field of research benefits from the impressive advancement of in-vivo exploration techniques of physiological parameters, imaging, genetics, and large-scale genomic approaches. The present review discusses the influence of mouse genetic on the most recent knowledge of the funny current's role in the physiology and pathophysiology of cardiac pacemaker activity. Genetically modified mice have provided important insights into the role of If in determining intrinsic automaticity in vivo and in myocytes of the conduction system. In addition, gene targeting of f-(HCN) channel isoforms have contributed to elucidating the current's role in the regulation of heart rate by the parasympathetic nervous system. This review is dedicated to Dario DiFrancesco on his retirement.

Use of Preoperative Single Dose Ivabradine for Perioperative Hemodynamic Stabilization During Non-Cardiac Elective Surgery Under General Anaesthesia: A Pilot Study.

BackgroundPeri-anesthetic hemodynamic fluctuations during non-cardiac surgeries are sometimes of serious consequences and associated with increased morbidity and mortality, especially in undiagnosed vulnerable patients. Currently used drugs like β-blocker, α2 agonist or sedative analgesic have their own limitations like combined negative ionotropic and chronotropic action, and unwanted bradycardia associated with hypotension, respectively. In this context, ivabradine has been used extensively in cases of cardiac failure, myocardial ischemia and cardiomyopathies for its funny channel associated dependable heart rate reducing property. Hence, for the first time, in search of a better agent for perioperative hemodynamic stabilization, the present study evaluated the role of ivabradine in patients undergoing non-cardiac surgeries.MethodsThis was a prospective, observer blind, randomized, interventional pilot study, conducted among 50 patients belonging undergoing elective abdominal laparoscopic surgeries, under general anesthesia. The study group patients received ivabradine tablet 7.5 mg, 2 h before scheduled time of surgery with a sip of water. All the patients received standardized balanced general anesthesia as practiced in our institute with all standard monitoring with additional minimum alveolar concentration (MAC) monitoring to ensure an adequate depth of anesthesia, and neuromuscular monitoring to ensure adequate and standard muscle relaxation. Hemodynamic stability of the groups was tested by comparing them at time points like induction, incision and operation, and extubation. Mean values of heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) were compared between groups, and also for variation with lapse of time, using RMANOVA analysis, using baseline parameters as covariate so as to standardize them.ResultsOn multivariate analysis using Wilk’s lambda multivariate test, there was a statistically significant difference (F = 3.587, P = 0.036) in HR between the groups with time, while no significant difference of SBP, DBP and MAP between the groups with time.ConclusionsThe study revealed a significant attenuation of HR response to stressful events like laryngoscopy, intubation and surgical incision with ivabradine. Also, a good intraoperative protection against cardiovascular ischemic and arrhythmic episodes in perioperative period was achieved with this drug ivabradine.

Discovery of Novel HCN4 Blockers with Unique Blocking Kinetics and Binding Properties.

The hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) channel underlies the pacemaker currents, called “If,” in sinoatrial nodes (SANs), which regulate heart rhythm. Some HCN4 blockers such as ivabradine have been extensively studied for treating various heart diseases. Studies have shown that these blockers have diverse state dependencies and binding sites, suggesting the existence of potential chemical and functional diversity among HCN4 blockers. Here we report approaches for the identification of novel HCN4 blockers through a random screening campaign among 16,000 small-molecule compounds using an automated patch-clamp system. These molecules exhibited various blockade profiles, and their blocking kinetics and associating amino acids were determined by electrophysiological studies and site-directed mutagenesis analysis, respectively. The profiles of these blockers were distinct from those of the previously reported HCN channel blockers ivabradine and ZD7288. Notably, the mutagenesis analysis showed that blockers with potencies that were increased when the channel was open involved a C478 residue, located at the pore cavity region near the cellular surface of the plasma membrane, while those with potencies that were decreased when the channel was open involved residues Y506 and I510, located at the intracellular region of the pore gate. Thus, this study reported for the first time the discovery of novel HCN4 blockers by screening, and their profiling analysis using an automated patch-clamp system provided chemical tools that will be useful to obtain unique molecular insights into the drug-binding modes of HCN4 and may contribute to the expansion of therapeutic options in the future.