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The mechanisms underlying heart automaticity are still poorly understood and controversial. Here we obtained, for the first time, complete conditional and time-controlled silencing of the hyperpolarization-activated "funny" current (If) by expression of a dominant-negative non-conductive human HCN4-channel subunit (hHCN4-AYA). Heart-specific If silencing recapitulated severe human disease of cardiac rhythm and conduction and showed that the functional role of f-channels in pacemaking critically depends on the activity of the autonomic nervous system. In line with this evidence, we were able to rescue failure of impulse generation and conduction by additional genetic deletion of cardiac muscarinic G-protein-activated (GIRK4) channels in If-deficient mice, without impairing heartbeat control. Our study establishes the role of f-channels in cardiac pacemaking and indicates that arrhythmia related to HCN loss-of-function in humans may be managed by pharmacological or genetic inhibition of GIRK4 channels, thus indicating a new unexplored therapeutic strategy to treat heart rhythm diseases. [ABSTRACT FROM AUTHOR]

Acute myocardial infarction (AMI) is the major cause of cardiovascular mortality worldwide. Early reperfusion is the only treatment recommended to reduce infarct size (IS). However, reperfusion presents also deleterious effects such as ischemia-reperfusion (IR) injury due to irreversible apoptotic death of cardiomyocytes. Most ischemic episodes are triggered by an increase in heart rate (HR) that induces an imbalance between myocardial oxygen delivery and consumption. The BEAUTIFUL clinical trial has demonstrated that moderate HR reduction diminishes the frequency of AMI episodes in patients with stable coronary artery disease and increased HR at rest. The HCN-mediated If current and the Cav1.3-mediated L-type Ca2+ currents play important roles in the generation of automaticity and HR, therefore they are interesting targets for selective control of HR and cardioprotection during AMI.The aim of our study was to investigate if targeting Cav1.3 channels could be an efficient strategy to reduce IS. Cav1.3 -/- mice was used as a genetic model of Cav1.3 inhibition because of the lack of selective blocker. Ivabradine, the selective f-channel blocker, was used for pure HR reduction as a positive control. Results show that selective HR decrease (40%) in an in vivo mouse model of acute MI is associated with reduced IR injury. Ivabradine administration 30 minutes before ischemia significantly reduced IS (35%). Cav1.3 -/- mice presented reduced IS (30%) compared to WT mice. In addition, preliminary results show that Girk4 -/- mice, a genetic model of moderate tachycardia (10%) displayed increased IS (45%) compared to control mice. In conclusion, results suggest a direct relationship between HR and IR injury and that inhibition of Cav1.3 channels constitutes a promising strategy to reduce both HR and IS. [ABSTRACT FROM AUTHOR]

The atrioventricular node (AVN) is a crucial component of the cardiac conduction system. Despite its pivotal role in regulating the transmission of electrical signals between atria and ventricles, a comprehensive understanding of the cellular electrophysiological mechanisms governing AVN function has remained elusive. This paper presents a detailed computational model of mouse AVN cell action potential (AP). Our model builds upon previous work and introduces several key refinements, including accurate representation of membrane currents and exchangers, calcium handling, cellular compartmentalization, dynamic update of intracellular ion concentrations, and calcium buffering. We recalibrated and validated the model against existing and unpublished experimental data. In control conditions, our model reproduces the AVN AP experimental features, (e.g. rate = 175 bpm, experimental range [121, 191] bpm). Notably, our study sheds light on the contribution of L-type calcium currents, through both Ca v 1.2 and Ca v 1.3 channels, in AVN cells. The model replicates several experimental observations, including the cessation of firing upon block of Ca v 1.3 or I Na,r current. I f block induces a reduction in beating rate of 11%. In summary, this work presents a comprehensive computational model of mouse AVN cell AP, offering a valuable tool for investigating pacemaking mechanisms and simulating the impact of ionic current blockades. By integrating calcium handling and refining formulation of ionic currents, our model advances understanding of this critical component of the cardiac conduction system, providing a platform for future developments in cardiac electrophysiology. KEY POINTS: This paper introduces a comprehensive computational model of mouse atrioventricular node (AVN) cell action potentials (APs). Our model is based on the electrophysiological data from isolated mouse AVN cells and exhibits an action potential and calcium transient that closely match the experimental records. By simulating the effects of blocking specific ionic currents, the model effectively predicts the roles of L-type Ca v 1.2 and Ca v 1.3 channels, T-type calcium channels, sodium currents (TTX-sensitive and TTX-resistant), and the funny current (I f ) in AVN pacemaking. The study also emphasizes the significance of other ionic currents, including I Kr , I to , I Kur , in regulating AP characteristics and cycle length in AVN cells. The model faithfully reproduces the rate dependence of action potentials under pacing, opening the possibility of use in impulse propagation models. The population-of-models approach showed the robustness of this new AP model in simulating a wide spectrum of cellular pacemaking in AVN., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Bradyarrhythmias including sinus bradycardia and atrioventricular (AV) block are frequently encountered in endurance athletes especially at night. While these are well tolerated by the young athlete, there is evidence that generally from the fifth decade of life onward, such arrhythmias can degenerate into pathological symptomatic bradycardia requiring pacemaker therapy. For many years, athletic bradycardia and AV block have been attributed to high vagal tone, but work from our group has questioned this widely held assumption and demonstrated a role for intrinsic electrophysiological remodeling of the sinus node and the AV node. In this article, we argue that bradyarrhythmias in the veteran athlete arise from the cumulative effects of exercise training, the circadian rhythm and aging on the electrical activity of the nodes. We consider contemporary strategies for the treatment of symptomatic bradyarrhythmias in athletes and highlight potential therapies resulting from our evolving mechanistic understanding of this phenomenon., Competing Interests: Disclosures The authors have no conflicts to disclose., (Copyright © 2024 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: Ventricular arrhythmias (VAs) demonstrate a prominent day-night rhythm, commonly presenting in the morning. Transcriptional rhythms in cardiac ion channels accompany this phenomenon, but their role in the morning vulnerability to VAs and the underlying mechanisms are not understood. We investigated the recruitment of transcription factors that underpins transcriptional rhythms in ion channels and assessed whether this mechanism was pertinent to the heart's intrinsic diurnal susceptibility to VA., Methods and Results: Assay for transposase-accessible chromatin with sequencing performed in mouse ventricular myocyte nuclei at the beginning of the animals' inactive (ZT0) and active (ZT12) periods revealed differentially accessible chromatin sites annotating to rhythmically transcribed ion channels and distinct transcription factor binding motifs in these regions. Notably, motif enrichment for the glucocorticoid receptor (GR; transcriptional effector of corticosteroid signaling) in open chromatin profiles at ZT12 was observed, in line with the well-recognized ZT12 peak in circulating corticosteroids. Molecular, electrophysiological, and in silico biophysically-detailed modeling approaches demonstrated GR-mediated transcriptional control of ion channels (including Scn5a underlying the cardiac Na + current, Kcnh2 underlying the rapid delayed rectifier K + current, and Gja1 responsible for electrical coupling) and their contribution to the day-night rhythm in the vulnerability to VA. Strikingly, both pharmacological block of GR and cardiomyocyte-specific genetic knockout of GR blunted or abolished ion channel expression rhythms and abolished the ZT12 susceptibility to pacing-induced VA in isolated hearts., Conclusions: Our study registers a day-night rhythm in chromatin accessibility that accompanies diurnal cycles in ventricular myocytes. Our approaches directly implicate the cardiac GR in the myocyte excitability rhythm and mechanistically link the ZT12 surge in glucocorticoids to intrinsic VA propensity at this time., Competing Interests: Disclosures None.

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes raise the possibility of generating pluripotent stem cells from a wide range of human diseases. In the cardiology field, hiPSCs have been used to address the mechanistic bases of primary arrhythmias and in investigations of drug safety. These studies have been focused primarily on atrial and ventricular pathologies. Consequently, many hiPSC-based cardiac differentiation protocols have been developed to differentiate between atrial- or ventricular-like cardiomyocytes. Few protocols have successfully proposed ways to obtain hiPSC-derived cardiac pacemaker cells, despite the very limited availability of human tissues from the sinoatrial node. Providing an in vitro source of pacemaker-like cells would be of paramount importance in terms of furthering our understanding of the mechanisms underlying sinoatrial node pathophysiology and testing innovative clinical strategies against sinoatrial node dysfunction (i.e., biological pacemakers and genetic- and pharmacological- based therapy). Here, we summarize and detail the currently available protocols used to obtain patient-derived pacemaker-like cells.

L-type voltage-gated calcium channels are involved in multiple physiological functions. Currently available antagonists do not discriminate between L-type channel isoforms. Importantly, no selective blocker is available to dissect the role of L-type isoforms Ca v 1.2 and Ca v 1.3 that are concomitantly co-expressed in the heart, neuroendocrine and neuronal cells. Here we show that calciseptine, a snake toxin purified from mamba venom, selectively blocks Ca v 1.2 -mediated L-type calcium currents (I CaL ) at concentrations leaving Ca v 1.3-mediated I CaL unaffected in both native cardiac myocytes and HEK-293T cells expressing recombinant Ca v 1.2 and Ca v 1.3 channels. Functionally, calciseptine potently inhibits cardiac contraction without altering the pacemaker activity in sino-atrial node cells, underscoring differential roles of Ca v 1.2- and Ca v 1.3 in cardiac contractility and automaticity. In summary, calciseptine is a selective L-type Ca v 1.2 Ca 2+ channel blocker and should be a valuable tool to dissect the role of these L-channel isoforms., (© 2024. The Author(s).)

Competing Interests: Disclosures M. De Waard is a founder and consultant for Smartox Biotechnology. The other authors report no conflicts.

Background: Acute myocardial infarction (AMI) is the major cause of cardiovascular mortality worldwide. Most ischemic episodes are triggered by an increase in heart rate, which induces an imbalance between myocardial oxygen delivery and consumption. Developing drugs that selectively reduce heart rate by inhibiting ion channels involved in heart rate control could provide more clinical benefits. The Ca v 1.3-mediated L-type Ca 2+ current ( I Cav1.3 ) play important roles in the generation of heart rate. Therefore, they can constitute relevant targets for selective control of heart rate and cardioprotection during AMI., Objective: We aimed to investigate the relationship between heart rate and infarct size using mouse strains knockout for Ca v 1.3 ( Ca v 1.3 -/- ) L-type calcium channel and of the cardiac G protein gated potassium channel ( Girk4 -/- ) in association with the funny (f)-channel inhibitor ivabradine., Methods: Wild-type (WT), Ca v 1.3 +/- , Ca v 1.3 -/- and Girk4 -/- mice were used as models of respectively normal heart rate, moderate heart rate reduction, bradycardia, and mild tachycardia, respectively. Mice underwent a surgical protocol of myocardial IR (40 min ischemia and 60 min reperfusion). Heart rate was recorded by one-lead surface ECG recording, and infarct size measured by triphenyl tetrazolium chloride staining. In addition, Ca v 1.3 -/- and WT hearts perfused on a Langendorff system were subjected to the same ischemia-reperfusion protocol ex vivo , without or with atrial pacing, and the coronary flow was recorded., Results: Ca v 1.3 -/- mice presented reduced infarct size (-29%), while Girk4 -/- displayed increased infarct size (+30%) compared to WT mice. Consistently, heart rate reduction in Ca v 1.3 +/- or by the f-channel blocker ivabradine was associated with significant decrease in infarct size (-27% and -32%, respectively) in comparison to WT mice., Conclusion: Our results show that decreasing heart rate allows to protect the myocardium against IR injury in vivo and reveal a close relationship between basal heart rate and IR injury. In addition, this study suggests that targeting Ca v 1.3 channels could constitute a relevant target for reducing infarct size, since maximal heart rate dependent cardioprotective effect is already observed in Ca v 1.3 +/- mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Delgado-Betancourt, Chinda, Mesirca, Barrere, Covinhes, Gallot, Vincent, Bidaud, Kumphune, Nargeot, Piot, Wickman, Mangoni and Barrère-Lemaire.)

Sepsis has emerged as a global health burden associated with multiple organ dysfunction and 20% mortality rate in patients. Numerous clinical studies over the past two decades have correlated the disease severity and mortality in septic patients with impaired heart rate variability (HRV), as a consequence of impaired chronotropic response of sinoatrial node (SAN) pacemaker activity to vagal/parasympathetic stimulation. However, the molecular mechanism(s) downstream to parasympathetic inputs have not been investigated yet in sepsis, particularly in the SAN. Based on electrocardiography, fluorescence Ca 2+ imaging, electrophysiology, and protein assays from organ to subcellular level, we report that impaired muscarinic receptor subtype 2-G protein-activated inwardly-rectifying potassium channel (M2R-GIRK) signaling in a lipopolysaccharide-induced proxy septic mouse model plays a critical role in SAN pacemaking and HRV. The parasympathetic responses to a muscarinic agonist, namely I KACh activation in SAN cells, reduction in Ca 2+ mobilization of SAN tissues, lowering of heart rate and increase in HRV, were profoundly attenuated upon lipopolysaccharide-induced sepsis. These functional alterations manifested as a direct consequence of reduced expression of key ion-channel components (GIRK1, GIRK4, and M2R) in the mouse SAN tissues and cells, which was further evident in the human right atrial appendages of septic patients and likely not mediated by the common proinflammatory cytokines elevated in sepsis.

Encoded by ANK2, ankyrin-B (AnkB) is a multifunctional adapter protein critical for the expression and targeting of key cardiac ion channels, transporters, cytoskeletal-associated proteins, and signaling molecules. Mice deficient for AnkB expression are neonatal lethal, and mice heterozygous for AnkB expression display cardiac structural and electrical phenotypes. Human ANK2 loss-of-function variants are associated with diverse cardiac manifestations; however, human clinical 'AnkB syndrome' displays incomplete penetrance. To date, animal models for human arrhythmias have generally been knock-out or transgenic overexpression models and thus the direct impact of ANK2 variants on cardiac structure and function in vivo is not clearly defined. Here, we directly tested the relationship of a single human ANK2 disease-associated variant with cardiac phenotypes utilizing a novel in vivo animal model. At baseline, young AnkBp.E1458G +/+ mice lacked significant structural or electrical abnormalities. However, aged AnkBp.E1458G +/+ mice displayed both electrical and structural phenotypes at baseline including bradycardia and aberrant heart rate variability, structural remodeling, and fibrosis. Young and old AnkBp.E1458G +/+ mice displayed ventricular arrhythmias following acute (adrenergic) stress. In addition, young AnkBp.E1458G +/+ mice displayed structural remodeling following chronic (transverse aortic constriction) stress. Finally, AnkBp.E1458G +/+ myocytes harbored alterations in expression and/or localization of key AnkB-associated partners, consistent with the underlying disease mechanism. In summary, our findings illustrate the critical role of AnkB in in vivo cardiac function as well as the impact of single AnkB loss-of-function variants in vivo. However, our findings illustrate the contribution and in fact necessity of secondary factors (aging, adrenergic challenge, pressure-overload) to phenotype penetrance and severity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Microcebus murinus, or gray mouse lemur (GML), is one of the smallest primates known, with a size in between mice and rats. The small size, genetic proximity to humans and prolonged senescence, make this lemur an emerging model for neurodegenerative diseases. For the same reasons, it could help understand how aging affects cardiac activity. Here, we provide the first characterization of sinoatrial (SAN) pacemaker activity and of the effect of aging on GML heart rate (HR). According to GML size, its heartbeat and intrinsic pacemaker frequencies lie in between those of mice and rats. To sustain this fast automaticity the GML SAN expresses funny and Ca 2+ currents (I f , I Ca,L and I Ca,T ) at densities similar to that of small rodents. SAN automaticity was also responsive to β-adrenergic and cholinergic pharmacological stimulation, showing a consequent shift in the localization of the origin of pacemaker activity. We found that aging causes decrease of basal HR and atrial remodeling in GML. We also estimated that, over 12 years of a lifetime, GML generates about 3 billion heartbeats, thus, as many as humans and three times more than rodents of equivalent size. In addition, we estimated that the high number of heartbeats per lifetime is a characteristic that distinguishes primates from rodents or other eutherian mammals, independently from body size. Thus, cardiac endurance could contribute to the exceptional longevity of GML and other primates, suggesting that GML's heart sustains a workload comparable to that of humans in a lifetime. In conclusion, despite the fast HR, GML replicates some of the cardiac deficiencies reported in old people, providing a suitable model to study heart rhythm impairment in aging. Moreover, we estimated that, along with humans and other primates, GML presents a remarkable cardiac longevity, enabling longer life span than other mammals of equivalent size., (© 2023. The Author(s).)

Background: Sinoatrial node cells (SANC) automaticity is generated by functional association between the activity of plasmalemmal ion channels and local diastolic intracellular Ca 2+ release (LCR) from ryanodine receptors. Strikingly, most isolated SANC exhibit a "dormant" state, whereas only a fraction shows regular firing as observed in intact SAN. Recent studies showed that β-adrenergic stimulation can initiate spontaneous firing in dormant SANC, though this mechanism is not entirely understood., Methods: To investigate the role of L-type Ca v 1.3 Ca 2+ channels in the adrenergic regulation of automaticity in dormant SANC, we used a knock-in mouse strain in which the sensitivity of L-type Ca v 1.2 α1 subunits to dihydropyridines (DHPs) was inactivated ( Ca v 1.2 DHP-/- ), enabling the selective pharmacological inhibition of Ca v 1.3 by DHPs., Results: In dormant SANC, β-adrenergic stimulation with isoproterenol (ISO) induced spontaneous action potentials (AP) and Ca 2+ transients, which were completely arrested with concomitant perfusion of the DHP nifedipine. In spontaneously firing SANC at baseline, Ca v 1.3 inhibition completely reversed the effect of β-adrenergic stimulation on AP and the frequency of Ca 2+ transients. Confocal calcium imaging of SANC showed that the β-adrenergic-induced synchronization of LCRs is regulated by the activity of Ca v 1.3 channels., Conclusions: Our study shows a novel role of Ca v 1.3 channels in initiating and maintaining automaticity in dormant SANC upon β-adrenergic stimulation.

Cardiac arrhythmias are a major cause of death and disability. A large number of experimental cell and animal models have been developed to study arrhythmogenic diseases. These models have provided important insights into the underlying arrhythmia mechanisms and translational options for their therapeutic management. This position paper from the ESC Working Group on Cardiac Cellular Electrophysiology provides an overview of (i) currently available in vitro, ex vivo, and in vivo electrophysiological research methodologies, (ii) the most commonly used experimental (cellular and animal) models for cardiac arrhythmias including relevant species differences, (iii) the use of human cardiac tissue, induced pluripotent stem cell (hiPSC)-derived and in silico models to study cardiac arrhythmias, and (iv) the availability, relevance, limitations, and opportunities of these cellular and animal models to recapitulate specific acquired and inherited arrhythmogenic diseases, including atrial fibrillation, heart failure, cardiomyopathy, myocarditis, sinus node, and conduction disorders and channelopathies. By promoting a better understanding of these models and their limitations, this position paper aims to improve the quality of basic research in cardiac electrophysiology, with the ultimate goal to facilitate the clinical translation and application of basic electrophysiological research findings on arrhythmia mechanisms and therapies., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Since its first description in 1979, the hyperpolarization-activated funny current (I f ) 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. I f 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 I f for more than 20 years. In 2001, the first study describing the electrophysiological properties of mouse sinoatrial pacemaker myocytes and those of I f 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 I f 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., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could be perceived to have influenced the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

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., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The understanding of the electrophysiological mechanisms that underlie mechanosensitivity of the sinoatrial node (SAN), the primary pacemaker of the heart, has been evolving over the past century. The heart is constantly exposed to a dynamic mechanical environment; as such, the SAN has numerous canonical and emerging mechanosensitive ion channels and signaling pathways that govern its ability to respond to both fast (within second or on beat-to-beat manner) and slow (minutes) timescales. This review summarizes the effects of mechanical loading on the SAN activity and reviews putative candidates, including fast mechanoactivated channels (Piezo, TREK, and BK) and slow mechanoresponsive ion channels [including volume-regulated chloride channels and transient receptor potential (TRP)], as well as the components of mechanochemical signal transduction, which may contribute to SAN mechanosensitivity. Furthermore, we examine the structural foundation for both mechano-electrical and mechanochemical signal transduction and discuss the role of specialized membrane nanodomains, namely, caveolae, in mechanical regulation of both membrane and calcium clock components of the so-called coupled-clock pacemaker system responsible for SAN automaticity. Finally, we emphasize how these mechanically activated changes contribute to the pathophysiology of SAN dysfunction and discuss controversial areas necessitating future investigations. Though the exact mechanisms of SAN mechanosensitivity are currently unknown, identification of such components, their impact into SAN pacemaking, and pathological remodeling may provide new therapeutic targets for the treatment of SAN dysfunction and associated rhythm abnormalities., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Turner, Kang, Mesirca, Hong, Mangoni, Glukhov and Sah.)

[Figure: see text].

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., (Copyright © 2020 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

The pacemaker cells of the cardiac sinoatrial node (SAN) are essential for normal cardiac automaticity. Dysfunction in cardiac pacemaking results in human sinoatrial node dysfunction (SND). SND more generally occurs in the elderly population and is associated with impaired pacemaker function causing abnormal heart rhythm. Individuals with SND have a variety of symptoms including sinus bradycardia, sinus arrest, SAN block, bradycardia/tachycardia syndrome, and syncope. Importantly, individuals with SND report chronotropic incompetence in response to stress and/or exercise. SND may be genetic or secondary to systemic or cardiovascular conditions. Current management of patients with SND is limited to the relief of arrhythmia symptoms and pacemaker implantation if indicated. Lack of effective therapeutic measures that target the underlying causes of SND renders management of these patients challenging due to its progressive nature and has highlighted a critical need to improve our understanding of its underlying mechanistic basis of SND. This review focuses on current information on the genetics underlying SND, followed by future implications of this knowledge in the management of individuals with SND., Competing Interests: The reviewer HZ declared a past co-authorship with the authors PM and MM to the handling editor. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wallace, El Refaey, Mesirca, Hund, Mangoni and Mohler.)

Background: Endurance athletes are prone to bradyarrhythmias, which in the long-term may underscore the increased incidence of pacemaker implantation reported in this population. Our previous work in rodent models has shown training-induced sinus bradycardia to be due to microRNA (miR)-mediated transcriptional remodeling of the HCN4 channel, leading to a reduction of the "funny" ( I f ) current in the sinoatrial node (SAN). Objective: To test if genetic ablation of G-protein-gated inwardly rectifying potassium channel, also known as I KACh channels prevents sinus bradycardia induced by intensive exercise training in mice. Methods: Control wild-type (WT) and mice lacking GIRK4 ( Girk4 -/- ), an integral subunit of I KACh were assigned to trained or sedentary groups. Mice in the trained group underwent 1-h exercise swimming twice a day for 28 days, 7 days per week. We performed electrocardiogram recordings and echocardiography in both groups at baseline, during and after the training period. At training cessation, mice were euthanized and SAN tissues were isolated for patch clamp recordings in isolated SAN cells and molecular profiling by quantitative PCR (qPCR) and western blotting. Results: At swimming cessation trained WT mice presented with a significantly lower resting HR that was reversible by acute I KACh block whereas Girk4 -/- mice failed to develop a training-induced sinus bradycardia. In line with HR reduction, action potential rate, density of I f , as well as of T- and L-type Ca 2+ currents ( I CaT and I CaL ) were significantly reduced only in SAN cells obtained from WT-trained mice. I f reduction in WT mice was concomitant with downregulation of HCN4 transcript and protein, attributable to increased expression of corresponding repressor microRNAs (miRs) whereas reduced I CaL in WT mice was associated with reduced Ca v 1.3 protein levels. Strikingly, I KACh ablation suppressed all training-induced molecular remodeling observed in WT mice. Conclusion: Genetic ablation of cardiac I KACh in mice prevents exercise-induced sinus bradycardia by suppressing training induced remodeling of inward currents I f , I CaT and I CaL due in part to the prevention of miR-mediated transcriptional remodeling of HCN4 and likely post transcriptional remodeling of Ca v 1.3. Strategies targeting cardiac I KACh may therefore represent an alternative to pacemaker implantation for bradyarrhythmias seen in some veteran athletes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bidaud, D’Souza, Forte, Torre, Greuet, Thirard, Anderson, Chung You Chong, Torrente, Roussel, Wickman, Boyett, Mangoni and Mesirca.)

The spontaneous activity of the sinoatrial node initiates the heartbeat. Sino-atrial node dysfunction (SND) and sick sinoatrial (sick sinus) syndrome are caused by the heart's inability to generate a normal sinoatrial node action potential. In clinical practice, SND is generally considered an age-related pathology, secondary to degenerative fibrosis of the heart pacemaker tissue. However, other forms of SND exist, including idiopathic primary SND, which is genetic, and forms that are secondary to cardiovascular or systemic disease. The incidence of SND in the general population is expected to increase over the next half century, boosting the need to implant electronic pacemakers. During the last two decades, our knowledge of sino-atrial node physiology and of the pathophysiological mechanisms underlying SND has advanced considerably. This review summarizes the current knowledge about SND mechanisms and discusses the possibility of introducing new pharmacologic therapies for treating SND.

Cardiac automaticity is set by pacemaker activity of the sinus node (SAN). In addition to the ubiquitously expressed cardiac voltage-gated L-type Ca v 1.2 Ca 2+ channel isoform, pacemaker cells within the SAN and the atrioventricular node co-express voltage-gated L-type Ca v 1.3 and T-type Ca v 3.1 Ca 2+ channels (SAN-VGCCs). The role of SAN-VGCCs in automaticity is incompletely understood. We used knockout mice carrying individual genetic ablation of Ca v 1.3 (Ca v 1.3 -/- ) or Ca v 3.1 (Ca v 3.1 -/- ) channels and double mutant Ca v 1.3 -/- /Ca v 3.1 -/- mice expressing only Ca v 1.2 channels. We show that concomitant loss of SAN-VGCCs prevents physiological SAN automaticity, blocks impulse conduction and compromises ventricular rhythmicity. Coexpression of SAN-VGCCs is necessary for impulse formation in the central SAN. In mice lacking SAN-VGCCs, residual pacemaker activity is predominantly generated in peripheral nodal and extranodal sites by f-channels and TTX-sensitive Na + channels. In beating SAN cells, ablation of SAN-VGCCs disrupted late diastolic local intracellular Ca 2+ release, which demonstrates an important role for these channels in supporting the sarcoplasmic reticulum based "Ca 2+ clock" mechanism during normal pacemaking. These data implicate an underappreciated role for co-expression of SAN-VGCCs in heart automaticity and define an integral role for these channels in mechanisms that control the heartbeat.

Ryanodine receptors are responsible for the massive release of calcium from the sarcoplasmic reticulum that triggers heart muscle contraction. Maurocalcin (MCa) is a 33 amino acid peptide toxin known to target skeletal ryanodine receptor. We investigated the effect of MCa and its analog MCaE12A on isolated cardiac ryanodine receptor (RyR2), and showed that they increase RyR2 sensitivity to cytoplasmic calcium concentrations promoting channel opening and decreases its sensitivity to inhibiting calcium concentrations. By measuring intracellular Ca2+ transients, calcium sparks and contraction on cardiomyocytes isolated from adult rats or differentiated from human-induced pluripotent stem cells, we demonstrated that MCaE12A passively penetrates cardiomyocytes and promotes the abnormal opening of RyR2. We also investigated the effect of MCaE12A on the pacemaker activity of sinus node cells from different mice lines and showed that, MCaE12A improves pacemaker activity of sinus node cells obtained from mice lacking L-type Cav1.3 channel, or following selective pharmacologic inhibition of calcium influx via Cav1.3. Our results identify MCaE12A as a high-affinity modulator of RyR2 and make it an important tool for RyR2 structure-to-function studies as well as for manipulating Ca2+ homeostasis and dynamic of cardiac cells., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

I Kr current, a major component of cardiac repolarization, is mediated by human Ether-à-go-go -Related Gene (hERG, K v 11.1) potassium channels. The blockage of these channels by pharmacological compounds is associated to drug-induced long QT syndrome (LQTS), which is a life-threatening disorder characterized by ventricular arrhythmias and defects in cardiac repolarization that can be illustrated using cardiomyocytes derived from human-induced pluripotent stem cells (hiPS-CMs). This study was meant to assess the modification in hiPS-CMs excitability and contractile properties by BeKm-1, a natural scorpion venom peptide that selectively interacts with the extracellular face of hERG, by opposition to reference compounds that act onto the intracellular face. Using an automated patch-clamp system, we compared the affinity of BeKm-1 for hERG channels with some reference compounds. We fully assessed its effects on the electrophysiological, calcium handling, and beating properties of hiPS-CMs. By delaying cardiomyocyte repolarization, the peptide induces early afterdepolarizations and reduces spontaneous action potentials, calcium transients, and contraction frequencies, therefore recapitulating several of the critical phenotype features associated with arrhythmic risk in drug-induced LQTS. BeKm-1 exemplifies an interesting reference compound in the integrated hiPS-CMs cell model for all drugs that may block the hERG channel from the outer face. Being a peptide that is easily modifiable, it will serve as an ideal molecular platform for the design of new hERG modulators displaying additional functionalities.

The above article was published online with an error in Fig. 1b. There is a doubled action potential at the far right of the left panel of the figure.

The heart automaticity is a fundamental physiological function in vertebrates. The cardiac impulse is generated in the sinus node by a specialized population of spontaneously active myocytes known as "pacemaker cells." Failure in generating or conducting spontaneous activity induces dysfunction in cardiac automaticity. Several families of ion channels are involved in the generation and regulation of the heart automaticity. Among those, voltage-gated L-type Cav1.3 (α1D) and T-type Cav3.1 (α1G) Ca 2+ channels play important roles in the spontaneous activity of pacemaker cells. Ca 2+ channel channelopathies specifically affecting cardiac automaticity are considered rare. Recent research on familial disease has identified mutations in the Cav1.3-encoding CACNA1D gene that underlie congenital sinus node dysfunction and deafness (OMIM # 614896). In addition, both Cav1.3 and Cav3.1 channels have been identified as pathophysiological targets of sinus node dysfunction and heart block, caused by congenital autoimmune disease of the cardiac conduction system. The discovery of channelopathies linked to Cav1.3 and Cav3.1 channels underscores the importance of Ca 2+ channels in the generation and regulation of heart's automaticity.

Sinus node (SAN) dysfunction (SND) manifests as low heart rate (HR) and is often accompanied by atrial tachycardia or atrioventricular (AV) block. The only currently available therapy for chronic SND is the implantation of an electronic pacemaker. Because of the growing burden of SND in the population, new pharmacological therapies of chronic SND and heart block are desirable. We developed a collection of genetically modified mouse strains recapitulating human primary SND associated with different degrees of AV block. These mice were generated with genetic ablation of L-type Ca v 1.3 (Ca v 1.3 -/- ), T-type Ca v 3.1 (Ca v 3.1 -/- ), or both (Ca v 1.3 -/- /Ca v 3.1 -/- ). We also studied mice haplo-insufficient for the Na + channel Na v 1.5 (Na v 1.5 +/ ) and mice in which the cAMP-dependent regulation of hyperpolarization-activated f-(HCN4) channels has been abolished (HCN4-CNBD). We analysed, by telemetric ECG recording, whether pharmacological inhibition of the G-protein-activated K + current (I KACh ) by the peptide tertiapin-Q could improve HR and AV conduction in these mouse strains. Tertiapin-Q significantly improved the HR of Ca v 1.3 -/- (19%), Ca v 1.3 -/- /Ca v 3.1 -/- (23%) and HCN4-CNBD (14%) mice. Tertiapin-Q also improved cardiac conduction of Na v 1.5 +/- mice by 24%. Our data suggest that the development of pharmacological I KACh inhibitors for the management of SND and conduction disease is a viable approach.

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 (TRIP8b nano ) that recapitulates affinity and gating effects of TRIP8b in HCN isoforms (hHCN1, mHCN2, rbHCN4) and in the cardiac current I f in rabbit and mouse sinoatrial node cardiomyocytes. Guided by an NMR-derived structural model that identifies the key molecular interactions between TRIP8b nano and the HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8b nano ) which successfully prevented β-adrenergic activation of mouse I f leaving the stimulation of the L-type calcium current (I CaL ) unaffected. TRIP8b nano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers., Competing Interests: AS, FC, AP, AB, DD, VM, CD, BI, PM, MM, GT, LB, BS, AM No competing interests declared, (© 2018, Saponaro et al.)

Competing Interests: Déclaration de liens d’intérêts Les auteurs déclarent ne pas avoir de liens d’intérêts.

Circadian clocks drive biological rhythms with a period of approximately 24 hours and keep in time with the outside world through daily resetting by environmental cues. While this external entrainment has been extensively investigated in the suprachiasmatic nuclei (SCN), the role of internal systemic rhythms, including daily fluctuations in core temperature or circulating hormones remains debated. Here, we show that lactating mice, which exhibit dampened systemic rhythms, possess normal molecular clockwork but impaired rhythms in both heat shock response gene expression and electrophysiological output in their SCN. This suggests that body rhythms regulate SCN activity downstream of the clock. Mathematical modeling predicts that systemic feedback upon the SCN functions as an internal oscillator that accounts for in vivo and ex vivo observations. Thus we are able to propose a new bottom-up hierarchical organization of circadian timekeeping in mammals, based on the interaction in the SCN between clock-dependent and system-driven oscillators.

The spontaneous activity of sinoatrial node (SAN) pacemaker cells is generated by a functional interplay between the activity of ionic currents of the plasma membrane and intracellular Ca 2+ dynamics. The molecular correlate of a dihydropyridine (DHP)-sensitive sustained inward Na + current (I st ), a key player in SAN automaticity, is still unknown. Here we show that I st and the L-type Ca 2+ current (I Ca,L ) share Ca V 1.3 as a common molecular determinant. Patch-clamp recordings of mouse SAN cells showed that I st is activated in the diastolic depolarization range, and displays Na + permeability and minimal inactivation and sensitivity to I Ca,L activators and blockers. Both Ca V 1.3-mediated I Ca,L and I st were abolished in Ca V 1.3-deficient (Ca V 1.3 -/- ) SAN cells but the Ca V 1.2-mediated I Ca,L current component was preserved. In SAN cells isolated from mice expressing DHP-insensitive Ca V 1.2 channels (Ca V 1.2 DHP-/- ), I st and Ca V 1.3-mediated I Ca,L displayed overlapping sensitivity and concentration-response relationships to the DHP blocker nifedipine. Consistent with the hypothesis that Ca V 1.3 rather than Ca V 1.2 underlies I st , a considerable fraction of I Ca,L was resistant to nifedipine inhibition in Ca V 1.2 DHP-/- SAN cells. These findings identify Ca V 1.3 channels as essential molecular components of the voltage-dependent, DHP-sensitive I st Na + current in the SAN.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a lethal genetic arrhythmia that manifests syncope or sudden death in children and young adults under stress conditions. CPVT patients often present bradycardia and sino-atrial node (SAN) dysfunction. However, the mechanism remains unclear. We analyzed SAN function in two CPVT families and in a novel knock-in (KI) mouse model carrying the RyR2R420Q mutation. Humans and KI mice presented slower resting heart rate. Accordingly, the rate of spontaneous intracellular Ca2+ ([Ca2+]i) transients was slower in KI mouse SAN preparations than in WT, without any significant alteration in the "funny" current (If ). The L-type Ca2+ current was reduced in KI SAN cells in a [Ca2+]i-dependent way, suggesting that bradycardia was due to disrupted crosstalk between the "voltage" and "Ca2+" clock, and the mechanisms of pacemaking was induced by aberrant spontaneous RyR2- dependent Ca2+ release. This finding was consistent with a higher Ca2+ leak during diastolic periods produced by long-lasting Ca2+ sparks in KI SAN cells. Our results uncover a mechanism for the CPVT-causing RyR2 N-terminal mutation R420Q, and they highlight the fact that enhancing the Ca2+ clock may slow the heart rhythm by disturbing the coupling between Ca2+ and voltage clocks.

Pacemaker activity of the sino-atrial node generates the heart rate. Disease of the sinus node and impairment of atrioventricular conduction induce an excessively low ventricular rate (bradycardia), which cannot meet the needs of the organism. Bradycardia accounts for about half of the total workload of clinical cardiologists. The 'sick sinus' syndrome (SSS) is characterized by sinus bradycardia and periods of intermittent atrial fibrillation. Several genetic or acquired risk factors or pathologies can lead to SSS. Implantation of an electronic pacemaker constitutes the only available therapy for SSS. The incidence of SSS is forecast to double over the next 50 years, with ageing of the general population thus urging the development of complementary or alternative therapeutic strategies. In recent years an increasing number of mutations affecting ion channels involved in sino-atrial automaticity have been reported to underlie inheritable SSS. L-type Ca v 1.3 channels play a major role in the generation and regulation of sino-atrial pacemaker activity and atrioventricular conduction. Mutation in the CACNA1D gene encoding Ca v 1.3 channels induces loss-of-function in channel activity and underlies the sino-atrial node dysfunction and deafness syndrome (SANDD). Mice lacking Ca v 1.3 channels (Ca v 1.3 -/- ) fairly recapitulate SSS and constitute a precious model to test new therapeutic approaches to handle this disease. Work in our laboratory shows that targeting G protein-gated K + (I KACh ) channels effectively rescues SSS of Ca v 1.3 -/- mice. This new concept of 'compensatory' ion channel targeting shines new light on the principles underlying the pacemaker mechanism and may open the way to new therapies for SSS., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Aims: Sino-atrial node (SAN) automaticity is an essential mechanism of heart rate generation that is still not completely understood. Recent studies highlighted the importance of intracellular Ca(2+) ([Ca(2+)]i) dynamics during SAN pacemaker activity. Nevertheless, the functional role of voltage-dependent L-type Ca(2+) channels in controlling SAN [Ca(2+)]i release is largely unexplored. Since Cav1.3 is the predominant L-type Ca(2+) channel isoform in SAN cells, we studied [Ca(2+)]i dynamics in isolated cells and ex vivo SAN preparations explanted from wild-type (WT) and Cav1.3 knockout (KO) mice (Cav1.3(-/-))., Methods and Results: We found that Cav1.3 deficiency strongly impaired [Ca(2+)]i dynamics, reducing the frequency of local [Ca(2+)]i release events and preventing their synchronization. This impairment inhibited the generation of Ca(2+) transients and delayed spontaneous activity. We also used action potentials recorded in WT SAN cells as voltage-clamp commands for Cav1.3(-/-) cells. Although these experiments showed abolished Ca(2+) entry through L-type Ca(2+) channels in the diastolic depolarization range of KO SAN cells, their sarcoplasmic reticulum Ca(2+) load remained normal. β-Adrenergic stimulation enhanced pacemaking of both genotypes, though, Cav1.3(-/-) SAN cells remained slower than WT. Conversely, we rescued pacemaker activity in Cav1.3(-/-) SAN cells and intact tissues through caffeine-mediated stimulation of Ca(2+)-induced Ca(2+) release., Conclusions: Cav1.3 channels play a critical role in the regulation of [Ca(2+)]i dynamics, providing an unanticipated mechanism for triggering local [Ca(2+)]i releases and thereby controlling pacemaker activity. Our study also provides an additional pathophysiological mechanism for congenital SAN dysfunction and heart block linked to Cav1.3 loss of function in humans., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.)

Dysfunction of pacemaker activity in the sinoatrial node (SAN) underlies "sick sinus" syndrome (SSS), a common clinical condition characterized by abnormally low heart rate (bradycardia). If untreated, SSS carries potentially life-threatening symptoms, such as syncope and end-stage organ hypoperfusion. The only currently available therapy for SSS consists of electronic pacemaker implantation. Mice lacking L-type Cav1.3 Ca(2+) channels (Cav1.3(-/-)) recapitulate several symptoms of SSS in humans, including bradycardia and atrioventricular (AV) dysfunction (heart block). Here, we tested whether genetic ablation or pharmacological inhibition of the muscarinic-gated K(+) channel (IKACh) could rescue SSS and heart block in Cav1.3(-/-) mice. We found that genetic inactivation of IKACh abolished SSS symptoms in Cav1.3(-/-) mice without reducing the relative degree of heart rate regulation. Rescuing of SAN and AV dysfunction could be obtained also by pharmacological inhibition of IKACh either in Cav1.3(-/-) mice or following selective inhibition of Cav1.3-mediated L-type Ca(2+) (ICa,L) current in vivo. Ablation of IKACh prevented dysfunction of SAN pacemaker activity by allowing net inward current to flow during the diastolic depolarization phase under cholinergic activation. Our data suggest that patients affected by SSS and heart block may benefit from IKACh suppression achieved by gene therapy or selective pharmacological inhibition.

Pacemaker activity of automatic cardiac myocytes controls the heartbeat in everyday life. Cardiac automaticity is under the control of several neurotransmitters and hormones and is constantly regulated by the autonomic nervous system to match the physiological needs of the organism. Several classes of ion channels and proteins involved in intracellular Ca(2+) dynamics contribute to pacemaker activity. The functional role of voltage-gated calcium channels (VGCCs) in heart automaticity and impulse conduction has been matter of debate for 30 years. However, growing evidence shows that VGCCs are important regulators of the pacemaker mechanisms and play also a major role in atrio-ventricular impulse conduction. Incidentally, studies performed in genetically modified mice lacking L-type Cav1.3 (Cav1.3(-/-)) or T-type Cav3.1 (Cav3.1(-/-)) channels show that genetic inactivation of these channels strongly impacts pacemaking. In cardiac pacemaker cells, VGCCs activate at negative voltages at the beginning of the diastolic depolarization and importantly contribute to this phase by supplying inward current. Loss-of-function of these channels also impairs atrio-ventricular conduction. Furthermore, inactivation of Cav1.3 channels promotes also atrial fibrillation and flutter in knockout mice suggesting that these channels can play a role in stabilizing atrial rhythm. Genomic analysis demonstrated that Cav1.3 and Cav3.1 channels are widely expressed in pacemaker tissue of mice, rabbits and humans. Importantly, human diseases of pacemaker activity such as congenital bradycardia and heart block have been attributed to loss-of-function of Cav1.3 and Cav3.1 channels. In this article, we will review the current knowledge on the role of VGCCs in the generation and regulation of heart rate and rhythm. We will discuss also how loss of Ca(2+) entry through VGCCs could influence intracellular Ca(2+) handling and promote atrial arrhythmias.

The mechanisms underlying cardiac automaticity are still incompletely understood and controversial. Here we report the complete conditional and time-controlled silencing of the 'funny' current (If) by expression of a dominant-negative, non-conductive HCN4-channel subunit (hHCN4-AYA). Heart-specific If silencing caused altered [Ca(2+)]i release and Ca(2+) handling in the sinoatrial node, impaired pacemaker activity and symptoms reminiscent of severe human disease of pacemaking. The effects of If silencing critically depended on the activity of the autonomic nervous system. We were able to rescue the failure of impulse generation and conduction by additional genetic deletion of cardiac muscarinic G-protein-activated (GIRK4) channels in If-deficient mice without impairing heartbeat regulation. Our study establishes the role of f-channels in cardiac automaticity and indicates that arrhythmia related to HCN loss-of-function may be managed by pharmacological or genetic inhibition of GIRK4 channels, thus offering a new therapeutic strategy for the treatment of heart rhythm diseases.