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Cardiac rhythms are related to heart electrical activity, being the essential aspect of the cardiovascular physiology. Usually, these rhythms are represented by electrocardiograms (ECGs) that are useful to detect cardiac pathologies. Essentially, the heart activity starts in the sinoatrial node (SA) node, the natural pacemaker, propagating to the atrioventricular node (AV), and finally reaching the His-Purkinje complex (HP). This paper investigates the control of cardiac rhythms in order to induce normal rhythms from pathological responses. A mathematical model that presents close agreement with experimental measurements is employed to represent the heart functioning. The adopted model comprises a network of three nonlinear oscillators that represent each one of the cardiac nodes, connected by delayed couplings. The pathological behavior is induced by an external stimulus in the SA node. An adaptive controller is proposed acting in the SA node considering an strategy based on the signal obtained by the natural pacemaker and its regularization. The incorporation of adaptive compensation in a Lyapunov-based control scheme allows the compensation for the unknown dynamics. The controller ability to deal with interpatient variability is evaluated by assuming that the heart model is not available to the controller design, being used only in the simulator to assess the control performance. Results show that the adaptive term can reduce the control effort by around 3% while reducing the tracking error by 20%, when compared to the conventional feedback approach. Additionally, the controller can avoid abnormal rhythms, turning the ECG closer to the expected normal behavior and preventing critical cardiac responses. Therefore, this work demonstrates that an adaptive controller can be used to regulate the ECG signal without prior information about the system and disregarding inter- and intrapatient variability., (© 2024. The Author(s).)

Aging is a major risk factor for sinoatrial node (SAN) dysfunction, which can impair heart rate (HR) control and heart rate variability (HRV). HR and HRV are determined by intrinsic SAN function and its regulation by the autonomic nervous system (ANS). The purpose of this study was to use multi-scale multi-fractal detrended fluctuation analysis (MSMFDFA; a complexity-based approach to analyze multi-fractal dynamics) to longitudinally assess changes in multi-fractal HRV properties and SAN function in ECG time series recorded repeatedly across the full adult lifespan in mice. ECGs were recorded in anesthetized mice in baseline conditions and after autonomic nervous system blockade every three months beginning at 6 months of age until the end of life. MSMFDFA was used to assess HRV and SAN function every three months between 6 and 27 months of age. Intrinsic HR (i.e. HR during ANS blockade) remained relatively stable until 15 months of age, and then progressively declined until study endpoint at 27 months of age. MSMFDFA revealed sudden and rapid changes in multi-fractal properties of the ECG RR interval time series in aging mice. In particular, multi-fractal spectrum width (MFSW, a measure of multi-fractality) was relatively stable between 6 months and 15 months of age and then progressively increased at 27 months of age. These changes in MFSW were evident in baseline conditions and during ANS blockade. Thus, intrinsic SAN function declines progressively during aging and is manifested by age-associated changes in multi-fractal HRV across the lifespan in mice, which can be accurately quantified by MSMFDFA., (© 2024. The Author(s), under exclusive licence to American Aging Association.)

Background and Objective . This study addresses the Force-Frequency relationship, a fundamental characteristic of cardiac muscle influenced by β 1 -adrenergic stimulation. This relationship reveals that heart rate (HR) changes at the sinoatrial node lead to alterations in ventricular cell contractility, increasing the force and decreasing relaxation time for higher beat rates. Traditional models lacking this relationship offer an incomplete physiological depiction, impacting the interpretation of in silico experiment results. To improve this, we propose a new mathematical model for ventricular myocytes, named 'Feed Forward Modeling' (FFM). Methods . FFM adjusts model parameters like channel conductance and Ca 2+ pump affinity according to stimulation frequency, in contrast to fixed parameter values. An empirical sigmoid curve guided the adaptation of each parameter, integrated into a rabbit ventricular cell electromechanical model. Model validation was achieved by comparing simulated data with experimental current-voltage (I-V) curves for L-type Calcium and slow Potassium currents. Results . FFM-enhanced simulations align more closely with physiological behaviors, accurately reflecting inotropic and lusitropic responses. For instance, action potential duration at 90% repolarization (APD90) decreased from 206 ms at 1 Hz to 173 ms at 4 Hz using FFM, contrary to the conventional model, where APD90 increased, limiting high-frequency heartbeats. Peak force also showed an increase with FFM, from 8.5 mN mm -2 at 1 Hz to 11.9 mN mm -2 at 4 Hz, while it barely changed without FFM. Relaxation time at 50% of maximum force (t 50 ) similarly improved, dropping from 114 ms at 1 Hz to 75.9 ms at 4 Hz with FFM, a change not observed without the model. Conclusion . The FFM approach offers computational efficiency, bypassing the need to model all beta-adrenergic pathways, thus facilitating large-scale simulations. The study recommends that frequency change experiments include fractional dosing of isoproterenol to better replicate heart conditions in vivo ., (© 2024 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

JGP study (Si et al. https://doi.org/10.1085/jgp.202413578) reveals that, although they are present at low levels and only generate small currents in the sinoatrial node, Kv1.1 channels have a significant impact on cardiac pacemaking., (© 2024 Rockefeller University Press.)

The heartbeat originates from spontaneous action potentials in specialized pacemaker cells within the sinoatrial node (SAN) of the right atrium. Voltage-gated potassium channels in SAN myocytes mediate outward K+ currents that regulate cardiac pacemaking by controlling action potential repolarization, influencing the time between heartbeats. Gene expression studies have identified transcripts for many types of voltage-gated potassium channels in the SAN, but most remain of unknown functional significance. One such gene is Kcna1, which encodes epilepsy-associated voltage-gated Kv1.1 K+ channel α-subunits that are important for regulating action potential firing in neurons and cardiomyocytes. Here, we investigated the functional contribution of Kv1.1 to cardiac pacemaking at the whole heart, SAN, and SAN myocyte levels by performing Langendorff-perfused isolated heart preparations, multielectrode array recordings, patch clamp electrophysiology, and immunocytochemistry using Kcna1 knockout (KO) and wild-type (WT) mice. Our results showed that either genetic or pharmacological ablation of Kv1.1 significantly decreased the SAN firing rate, primarily by impairing SAN myocyte action potential repolarization. Voltage-clamp electrophysiology and immunocytochemistry revealed that Kv1.1 exerts its effects despite contributing only a small outward K+ current component, which we term IKv1.1, and despite apparently being present in low abundance at the protein level in SAN myocytes. These findings establish Kv1.1 as the first identified member of the Kv1 channel family to play a role in sinoatrial function, thereby rendering it a potential candidate and therapeutic targeting of sinus node dysfunction. Furthermore, our results demonstrate that small currents generated via low-abundance channels can still have significant impacts on cardiac pacemaking., (© 2024 Si et al.)

Loss or dysregulation of the normally precise control of heart rate via the autonomic nervous system plays a critical role during the development and progression of cardiovascular disease-including ischemic heart disease, heart failure, and arrhythmias. While the clinical significance of regulating changes in heart rate, known as the chronotropic effect, is undeniable, the mechanisms controlling these changes remain not fully understood. Heart rate acceleration and deceleration are mediated by increasing or decreasing the spontaneous firing rate of pacemaker cells in the sinoatrial node. During the transition from rest to activity, sympathetic neurons stimulate these cells by activating β-adrenergic receptors and increasing intracellular cyclic adenosine monophosphate. The same signal transduction pathway is targeted by positive chronotropic drugs such as norepinephrine and dobutamine, which are used in the treatment of cardiogenic shock and severe heart failure. The cyclic adenosine monophosphate-sensitive hyperpolarization-activated current (I f ) in pacemaker cells is passed by hyperpolarization-activated cyclic nucleotide-gated cation channels and is critical for generating the autonomous heartbeat. In addition, this current has been suggested to play a central role in the chronotropic effect. Recent studies demonstrate that cyclic adenosine monophosphate-dependent regulation of HCN4 (hyperpolarization-activated cyclic nucleotide-gated cation channel isoform 4) acts to stabilize the heart rate, particularly during rapid rate transitions induced by the autonomic nervous system. The mechanism is based on creating a balance between firing and recently discovered nonfiring pacemaker cells in the sinoatrial node. In this way, hyperpolarization-activated cyclic nucleotide-gated cation channels may protect the heart from sinoatrial node dysfunction, secondary arrhythmia of the atria, and potentially fatal tachyarrhythmia of the ventricles. Here, we review the latest findings on sinoatrial node automaticity and discuss the physiological and pathophysiological role of HCN pacemaker channels in the chronotropic response and beyond., Competing Interests: Disclosures None.

Strong circadian (~24h) rhythms in heart rate (HR) are critical for flexible regulation of cardiac pacemaking function throughout the day. While this circadian flexibility in HR is sustained in diverse conditions, it declines with age, accompanied by reduced maximal HR performance. The intricate regulation of circadian HR involves the orchestration of the autonomic nervous system (ANS), circadian rhythms of body temperature (CRBT), and local circadian rhythmicity (LCR), which has not been fully understood. Here, we developed a mathematical model describing ANS, CRBT, and LCR in sinoatrial nodal cells (SANC) that accurately captures distinct circadian patterns in adult and aged mice. Our model underscores how the alliance among ANS, CRBT, and LCR achieves circadian flexibility to cover a wide range of firing rates in SANC, performance to achieve maximal firing rates, while preserving robustness to generate rhythmic firing patterns irrespective of external conditions. Specifically, while ANS dominates in promoting SANC flexibility and performance, CRBT and LCR act as primary and secondary boosters, respectively, to further enhance SANC flexibility and performance. Disruption of this alliance with age results in impaired SANC flexibility and performance, but not robustness. This unexpected outcome is primarily attributed to the age-related reduction in parasympathetic activities, which maintains SANC robustness while compromising flexibility. Our work sheds light on the critical alliance of ANS, CRBT, and LCR in regulating time-of-day cardiac pacemaking function and dysfunction, offering insights into novel therapeutic targets for the prevention and treatment of cardiac arrhythmias., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Li, Kim. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The electrical impulses that coordinate the sequential, rhythmic contractions of the atria and ventricles are initiated and tightly regulated by the specialized tissues of the cardiac conduction system. In the mature heart, these impulses are generated by the pacemaker cardiomyocytes of the sinoatrial node, propagated through the atria to the atrioventricular node where they are delayed and then rapidly propagated to the atrioventricular bundle, right and left bundle branches, and finally, the peripheral ventricular conduction system. Each of these specialized components arise by complex patterning events during embryonic development. This chapter addresses the origins and transcriptional networks and signaling pathways that drive the development and maintain the function of the cardiac conduction system., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The sinoatrial node (SAN), the primary pacemaker of the heart, is responsible for the initiation and robust regulation of sinus rhythm. 3D mapping studies of the ex-vivo human heart suggested that the robust regulation of sinus rhythm relies on specialized fibrotically-insulated pacemaker compartments (head, center and tail) with heterogeneous expressions of key ion channels and receptors. They also revealed up to five sinoatrial conduction pathways (SACPs), which electrically connect the SAN with neighboring right atrium (RA). To elucidate the role of these structural-molecular factors in the functional robustness of human SAN, we developed comprehensive biophysical computer models of the SAN based on 3D structural, functional and molecular mapping of ex-vivo human hearts. Our key finding is that the electrical insulation of the SAN except SACPs, the heterogeneous expression of If, INa currents and adenosine A1 receptors (A1R) across SAN pacemaker-conduction compartments are required to experimentally reproduce observed SAN activation patterns and important phenomena such as shifts of the leading pacemaker and preferential SACP. In particular, we found that the insulating border between the SAN and RA, is required for robust SAN function and protection from SAN arrest during adenosine challenge. The heterogeneity in the expression of A1R within the human SAN compartments underlies the direction of pacemaker shift and preferential SACPs in the presence of adenosine. Alterations of INa current and fibrotic remodelling in SACPs can significantly modulate SAN conduction and shift the preferential SACP/exit from SAN. Finally, we show that disease-induced fibrotic remodeling, INa suppression or increased adenosine make the human SAN vulnerable to pacing-induced exit blocks and reentrant arrhythmia. In summary, our computer model recapitulates the structural and functional features of the human SAN and can be a valuable tool for investigating mechanisms of SAN automaticity and conduction as well as SAN arrhythmia mechanisms under different pathophysiological conditions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Sinoatrial node (SAN) beating interval variability (BIV) and the average beating interval (BI) are regulated by a coupled-clock system, driven by Ca 2+ -calmodulin activated adenylyl cyclase, cAMP, and downstream PKA signaling. Reduced responsiveness of the BI and BIV to submaximal, [X] 50 , β-adrenergic receptor (β-AR) stimulation, and phosphodiesterase inhibition (PDEI) have been documented in aged SAN tissue, whereas the maximal responses, [X] max , do not differ by age. To determine whether age-associated dysfunction in cAMP signaling leads to altered responsiveness of BI and BIV, we measured cAMP levels and BI in adult (2-4 months n = 27) and aged (22-26 months n = 25) C57/BL6 mouse SAN tissue in control and in response to β-AR or PDEI at X 50 and [X] max . Both cAMP and average BI in adult SAN were reduced at X 50 , whereas cAMP and BI at X max did not differ by age. cAMP levels and average BI were correlated both within and between adult and aged SAN. BIV parameters in long- and short-range terms were correlated with cAMP levels for adult SAN. However, due to reduced cAMP within aged tissues at [X] 50 , these correlations were diminished in advanced age. Thus, cAMP level generated by the coupled clock mechanisms is tightly linked to average BI. Reduced cAMP level at X 50 in aged SAN explains the reduced responsiveness of the BI and BIV to β-AR stimulation and PDEI., (© 2023. The Author(s), under exclusive licence to American Aging Association.)

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.

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system 1 . The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug-target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG + and IgA + plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs., (© 2023. The Author(s).)

Although, for many decades, the day-night rhythm in resting heart rate has been attributed to the parasympathetic branch of the autonomic nervous system (high vagal tone during sleep), recently we have shown that there is a circadian clock in the cardiac pacemaker, the sinus node, and the day-night rhythm in heart rate involves an intrinsic rhythmic transcriptional remodelling of pacemaker ion channels, particularly Hcn4 . We have now investigated the role of the sympathetic branch of the autonomic nervous system in this and shown it to have a non-canonical role. In mice, sustained long-term block of cardiac β-adrenergic receptors by propranolol administered in the drinking water abolished the day-night rhythm in pacemaking in the isolated sinus node. Concomitant with this, there was a loss of the normal day-night rhythm in many pacemaker ion channel transcripts. However, there was little or no change in the local circadian clock, indicating that the well-known day-night rhythm in sympathetic nerve activity is directly involved in pacemaker ion channel transcription. The day-night rhythm in pacemaking helps explain the occurrence of clinically significant bradyarrhythmias during sleep, and this study improves our understanding of this pathology. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

The sinoatrial node (SAN) is the primary pacemaker of the mammalian heart, initiating its electrical activation and ensuring that the heart's functional cardiac output meets physiological demand. SAN dysfunction (SND) can cause complex cardiac arrhythmias that can manifest as severe sinus bradycardia, sinus arrest, chronotropic incompetence and increased susceptibility to atrial fibrillation, among other cardiac conditions. SND has a complex aetiology, with both pre-existing disease and heritable genetic variation predisposing individuals to this pathology. In this Review, we summarize the current understanding of the genetic contributions to SND and the insights that they provide into this disorder's underlying molecular mechanisms. With an improved understanding of these molecular mechanisms, we can improve treatment options for SND patients and develop new therapeutics., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Cardiac pacemaker cells (CPCs) initiate the electric impulses that drive the rhythmic beating of the heart. CPCs reside in a heterogeneous, ECM-rich microenvironment termed the sinoatrial node (SAN). Surprisingly, little is known regarding the biochemical composition or mechanical properties of the SAN, and how the unique structural characteristics present in this region of the heart influence CPC function remains poorly understood. Here, we have identified that SAN development involves the construction of a "soft" macromolecular ECM that specifically encapsulates CPCs. In addition, we demonstrate that subjecting embryonic CPCs to substrate stiffnesses higher than those measured in vivo results in loss of coherent electrical oscillation and dysregulation of the HCN4 and NCX1 ion channels required for CPC automaticity. Collectively, these data indicate that local mechanics play a critical role in maintaining the embryonic CPC function while also quantitatively defining the range of material properties that are optimal for embryonic CPC maturation., (© 2023 Henley et al.)

Histological identification of the human sinoatrial node (SAN) remains a challenge. Conventional identification methods, such as Lev's method, have certain limitations. The aim of our study was to develop a new histological identification method that could properly identify the sinoatrial node, applicable to the immunohistochemical study of intra-nodal structures. Thirty-nine human autopsied hearts were included in this study. The cases included 23 men and 16 women ranging in age from 20 to 99 years. The sinoatrial area from eight control samples was cut in the vertical section using the conventional Lev's method. In our new method, called the "En face one-block method," the sinoatrial node was cut in "En face" at the junction of the right border of the right appendage and superior vena cava, placed in one long cassette, and serially cut using a microtome. Immunostaining was performed using primary antibodies against CD31, podoplanin (D2-40), S-100, and other proteins. The average area of the SAN on the slide glass in our new method was 32.2 mm 2 , which was significantly larger than that (3.59 mm 2 ) of the control samples by Lev's method. The SAN area was positively correlated with age (r = 0.357; p = 0.026), especially in women (r = 0.626; p = 0.0095). The SAN group had significantly lower percentage of CD31-positive blood capillaries, higher percentage of podoplanin-positive lymphatic channels, and S-100-positive peripheral nerves. We successfully developed a novel cutting method applicable to immunohistochemical studies, with which we could provide a bird's-eye view of the sinoatrial nodes., (© 2022. The Author(s), under exclusive licence to Japanese Association of Anatomists.)

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used to screen and characterize drugs and to reveal mechanisms underlying cardiac diseases. However, before hiPSC-CMs can be used as a reliable experimental model, the physiological mechanisms underlying their normal function should be further explored. Accordingly, a major feature of hiPSC-CMs is automaticity, which is regulated by both Ca2+ and membrane clocks. To investigate the mechanisms coupling these clocks, we tested three hypotheses: (1) normal automaticity of spontaneously beating hiPSC-CMs is regulated by local Ca2+ releases (LCRs) and cAMP/PKA-dependent coupling of Ca2+ clock to M clock; (2) the LCR period indicates the level of crosstalk within the coupled-clock system; and (3) perturbing the activity of even one clock can lead to hiPSC-CM-altered automaticity due to diminished crosstalk within the coupled-clock system. By measuring the local and global Ca2+ transients, we found that the LCRs properties are correlated with the spontaneous beat interval. Changes in cAMP-dependent coupling of the Ca2+ and M clocks, caused by a pharmacological intervention that either activates the β-adrenergic or cholinergic receptor or upregulates/downregulates PKA signaling, affected LCR properties, which in turn altered hiPSC-CMs automaticity. Clocks' uncoupling by attenuating the pacemaker current If or the sarcoplasmic reticulum Ca2+ kinetics, decreased hiPSC-CMs beating rate, and prolonged the LCR period. Finally, LCR characteristics of spontaneously beating (at comparable rates) hiPSC-CMs and rabbit SAN are similar. In conclusion, hiPSC-CM automaticity is controlled by the coupled-clock system whose function is mediated by Ca2+-cAMP-PKA signaling., (© 2022 Mazgaoker et al.)

The primary pacemaking activity of the heart is determined by a spontaneous action potential (AP) within sinoatrial node (SAN) cells. This unique AP generation relies on two mechanisms: membrane clocks and calcium clocks. Nonhomologous arrhythmias are caused by several functional and structural changes in the myocardium. MicroRNAs (miRNAs) are essential regulators of gene expression in cardiomyocytes. These miRNAs play a vital role in regulating the stability of cardiac conduction and in the remodeling process that leads to arrhythmias. Although it remains unclear how miRNAs regulate the expression and function of ion channels in the heart, these regulatory mechanisms may support the development of emerging therapies. This study discusses the spread and generation of AP in the SAN as well as the regulation of miRNAs and individual ion channels. Arrhythmogenicity studies on ion channels will provide a research basis for miRNA modulation as a new therapeutic target., Competing Interests: Declaration of competing interest This article had not been published previously and is not under consideration for publication elsewhere. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, and all authors approved the publication. The authors declared that it would not be published elsewhere in the same form, in English or any other language, including electronically, without the copyright holder's written consent if this article is accepted., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The sympathetic nervous system fight-or-flight response is characterized by a rapid increase in heart rate, which is mediated by an increase in the spontaneous action potential (AP) firing rate of pacemaker cells in the sinoatrial node. Sympathetic neurons stimulate sinoatrial myocytes (SAMs) by activating β adrenergic receptors (βARs) and increasing cAMP. The funny current (If) is among the cAMP-sensitive currents in SAMs. If is critical for pacemaker activity, however, its role in the fight-or-flight response remains controversial. In this study, we used AP waveform analysis, machine learning, and dynamic clamp experiments in acutely isolated SAMs from mice to quantitatively define the AP waveform changes and role of If in the fight-or-flight increase in AP firing rate. We found that while βAR stimulation significantly altered nearly all AP waveform parameters, the increase in firing rate was only correlated with changes in a subset of parameters (diastolic duration, late AP duration, and diastolic depolarization rate). Dynamic clamp injection of the βAR-sensitive component of If showed that it accounts for ∼41% of the fight-or-flight increase in AP firing rate and 60% of the decrease in the interval between APs. Thus, If is an essential contributor to the fight-or-flight increase in heart rate., (© 2022 Peters et al.)

The combined influences of sinoatrial nodal (SAN) pacemaker cell automaticity and its response to autonomic input determine the heart's beating interval variability and mean beating rate. To determine the intrinsic SAN and autonomic signatures buried within EKG RR interval time series change in advanced age, we measured RR interval variability before and during double autonomic blockade at 3-month intervals from 6 months of age until the end of life in long-lived (those that achieved the total cohort median life span of 24 months and beyond) C57/BL6 mice. Prior to 21 months of age, time-dependent changes in intrinsic RR interval variability and mean RR interval were relatively minor. Between 21 and 30 months of age, however, marked changes emerged in intrinsic SAN RR interval variability signatures, pointing to a reduction in the kinetics of pacemaker clock mechanisms, leading to reduced synchronization of molecular functions within and among SAN cells. This loss of high-frequency signal processing within intrinsic SAN signatures resulted in a marked increase in the mean intrinsic RR interval. The impact of autonomic signatures on RR interval variability were net sympathetic and partially compensated for the reduced kinetics of the intrinsic SAN RR interval variability signatures, and partially, but not completely, shifted the EKG RR time series intervals to a more youthful pattern. Cross-sectional analyses of other subsets of C57/BL6 ages indicated that at or beyond the median life span of our longitudinal cohort, noncardiac, constitutional, whole-body frailty was increased, energetic efficiency was reduced, and the respiratory exchange ratio increased. We interpret the progressive reduction in kinetics in intrinsic SAN RR interval variability signatures in this context of whole-body frailty beyond 21 months of age to be a manifestation of "heartbeat frailty.", (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Excitation-contraction coupling kinetics is dictated by the action potential rate of sinoatrial-nodal cells. These cells generate local Ca releases (LCRs) that activate Na/Ca exchanger current, which accelerates diastolic depolarization and determines the pace. LCRs are generated by clusters of ryanodine receptors, Ca release units (CRUs), residing in the sarcoplasmic reticulum. While CRU distribution exhibits substantial heterogeneity, its functional importance remains unknown. Using numerical modeling, here we show that with a square lattice distribution of CRUs, Ca-induced-Ca-release propagation during diastolic depolarization is insufficient for pacemaking within a broad range of realistic ICaL densities. Allowing each CRU to deviate randomly from its lattice position allows sparks to propagate, as observed experimentally. As disorder increases, the CRU distribution exhibits larger empty spaces and simultaneously CRU clusters, as in Poisson clumping. Propagating within the clusters, Ca release becomes synchronized, increasing action potential rate and reviving pacemaker function of dormant/nonfiring cells. However, cells with fully disordered CRU positions could not reach low firing rates and their β-adrenergic-receptor stimulation effect was substantially decreased. Inclusion of Cav1.3, a low-voltage activation L-type Ca channel isoform into ICaL, strongly increases recruitment of CRUs to fire during diastolic depolarization, increasing robustness of pacemaking and complementing effects of CRU distribution. Thus, order/disorder in CRU locations along with Cav1.3 expression regulates pacemaker function via synchronization of CRU firing. Excessive CRU disorder and/or overexpression of Cav1.3 boosts pacemaker function in the basal state, but limits the rate range, which may contribute to heart rate range decline with age and disease., (© 2022 Maltsev et al.)

The sinoatrial node (SAN) is composed of highly specialized cells that mandate the spontaneous beating of the heart through self-generation of an action potential (AP). Despite this automaticity, the SAN is under the modulation of the autonomic nervous system (ANS). In diabetes mellitus (DM), heart rate variability (HRV) manifests as a hallmark of diabetic cardiomyopathy. This is paralleled by an impaired regulation of the ANS, and by a pathological remodeling of the pacemaker structure and function. The direct effect of diabetes on the molecular signatures underscoring this pathology remains ill-defined. The recent focus on the electrical currents of the SAN in diabetes revealed a repressed firing rate of the AP and an elongation of its tracing, along with conduction abnormalities and contractile failure. These changes are blamed on the decreased expression of ion transporters and cell-cell communication ports at the SAN (i.e., HCN4, calcium and potassium channels, connexins 40, 45, and 46) which further promotes arrhythmias. Molecular analysis crystallized the RGS4 (regulator of potassium currents), mitochondrial thioredoxin-2 (reactive oxygen species; ROS scavenger), and the calcium-dependent calmodulin kinase II (CaMKII) as metabolic culprits of relaying the pathological remodeling of the SAN cells (SANCs) structure and function. A special attention is given to the oxidation of CaMKII and the generation of ROS that induce cell damage and apoptosis of diabetic SANCs. Consequently, the diabetic SAN contains a reduced number of cells with significant infiltration of fibrotic tissues that further delay the conduction of the AP between the SANCs. Failure of a genuine generation of AP and conduction of their derivative waves to the neighboring atrial myocardium may also occur as a result of the anti-diabetic regiment (both acute and/or chronic treatments). All together, these changes pose a challenge in the field of cardiology and call for further investigations to understand the etiology of the structural/functional remodeling of the SANCs in diabetes. Such an understanding may lead to more adequate therapies that can optimize glycemic control and improve health-related outcomes in patients with diabetes., 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 © 2022 Al Kury, Chacar, Alefishat, Khraibi and Nader.)

Heart rate (HR) is controlled by the sinoatrial node (SAN). SAN dysfunction is highly prevalent in aging; however, not all individuals age at the same rate. Rather, health status during aging is affected by frailty. Natriuretic peptides regulate SAN function in part by activating natriuretic peptide receptor C (NPR-C). The impacts of NPR-C on HR and SAN function in aging and as a function of frailty are unknown. Frailty was measured in aging wild-type and NPR-C knockout (NPR-C-/-) mice using a mouse clinical frailty index (FI). HR and SAN structure and function were investigated using intracardiac electrophysiology in anesthetized mice, high-resolution optical mapping in intact atrial preparations, histology, and molecular biology. NPR-C-/- mice rapidly became frail leading to shortened life span. HR was reduced and SAN recovery time was increased in older versus younger mice, and these changes were exacerbated in NPR-C-/- mice; however, there was substantial variability among age groups and genotypes. HR and SAN recovery time were correlated with FI score and fell along a continuum regardless of age or genotype. Optical mapping demonstrates impairments in SAN function that were also correlated with FI score. SAN fibrosis was increased in aged and NPR-C-/- mice and was graded by FI score. Loss of NPR-C results in accelerated aging and rapid decline in health status in association with impairments in HR and SAN function. Frailty assessment was effective and better able to distinguish aging-dependent changes in SAN function in the setting of shortened life span due to loss of NPR-C., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The mouse is a common and cost-effective animal model for basic research, and the number of genetically engineered mouse models with cardiac phenotype is increasing. In vivo electrophysiological study in mice is similar to that performed in humans. It is indispensable for acquiring intracardiac electrocardiogram recordings and determining baseline cardiac cycle intervals. Furthermore, the use of programmed electrical stimulation enables determination of parameters such as sinoatrial conduction time, sinus node recovery time, atrioventricular-nodal conduction properties, Wenckebach periodicity, refractory periods and arrhythmia vulnerability. This protocol describes specific procedures for determining these parameters that were adapted from analogous human protocols for use in mice. We include details of ex vivo electrophysiological study, which provides detailed insights into intrinsic cardiac electrophysiology without external influences from humoral and neural factors. In addition, we describe a heart preparation with intact innervation by the vagus nerve that can be used as an ex vivo model for vagal control of the cardiac conduction system. Data acquisition for in vivo and ex vivo electrophysiological study takes ~1 h per mouse, depending on the number of stimulation protocols applied during the procedure. The technique yields highly reliable results and can be used for phenotyping of cardiac disease models, elucidating disease mechanisms and confirming functional improvements in gene therapy approaches as well as for drug and toxicity testing., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Simplified nonlinear models of biological cells are widely used in computational electrophysiology. The models reproduce qualitatively many of the characteristics of various organs, such as the heart, brain, and intestine. In contrast to complex cellular ion-channel models, the simplified models usually contain a small number of variables and parameters, which facilitates nonlinear analysis and reduces computational load. In this paper, we consider pacemaking variants of the Aliev-Panfilov and Corrado two-variable excitable cell models. We conducted a numerical simulation study of these models and investigated the main nonlinear dynamic features of both isolated cells and 1D coupled pacemaker-excitable systems. Simulations of the 2D sinoatrial node and 3D intestine tissue as application examples of combined pacemaker-excitable systems demonstrated results similar to obtained previously. The uniform formulation for the conventional excitable cell models and proposed pacemaker models allows a convenient and easy implementation for the construction of personalized physiological models, inverse tissue modeling, and development of real-time simulation systems for various organs that contain both pacemaker and excitable cells., Competing Interests: The authors have declared that no competing interests exist.

The cardiac pacemaker ignites and coordinates the contraction of the whole heart, uninterruptedly, throughout our entire life. Pacemaker rate is constantly tuned by the autonomous nervous system to maintain body homeostasis. Sympathetic and parasympathetic terminals act over the pacemaker cells as the accelerator and the brake pedals, increasing or reducing the firing rate of pacemaker cells to match physiological demands. Despite the remarkable reliability of this tissue, the pacemaker is not exempt from the detrimental effects of aging. Mammals experience a natural and continuous decrease in the pacemaker rate throughout the entire lifespan. Why the pacemaker rhythm slows with age is poorly understood. Neural control of the pacemaker is remodeled from birth to adulthood, with strong evidence of age-related dysfunction that leads to a downshift of the pacemaker. Such evidence includes remodeling of pacemaker tissue architecture, alterations in the innervation, changes in the sympathetic acceleration and the parasympathetic deceleration, and alterations in the responsiveness of pacemaker cells to adrenergic and cholinergic modulation. In this review, we revisit the main evidence on the neural control of the pacemaker at the tissue and cellular level and the effects of aging on shaping this neural control., (© 2021. The Author(s).)

The heartbeat is initiated by pacemaker cells residing in the sinoatrial node (SAN). SAN cells generate spontaneous action potentials (APs), i.e., normal automaticity. The sympathetic nervous system increases the heart rate commensurate with the cardiac output demand via stimulation of SAN β-adrenergic receptors (βAR). While SAN cells reportedly represent a highly heterogeneous cell population, the current dogma is that, in response to βAR stimulation, all cells increase their spontaneous AP firing rate in a similar fashion. The aim of the present study was to investigate the cell-to-cell variability in the responses of a large population of SAN cells. We measured the βAR responses among 166 single SAN cells isolated from 33 guinea pig hearts. In contrast to the current dogma, the SAN cell responses to βAR stimulation substantially varied. In each cell, changes in the AP cycle length were highly correlated (R 2 = 0.97) with the AP cycle length before βAR stimulation. While, as expected, on average, the cells increased their pacemaker rate, greater responses were observed in cells with slower basal rates, and vice versa: cells with higher basal rates showed smaller responses, no responses, or even decreased their rate. Thus, βAR stimulation synchronized the operation of the SAN cell population toward a higher average rate, rather than uniformly shifting the rate in each cell, creating a new paradigm of βAR-driven fight-or-flight responses among individual pacemaker cells.

Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current (I f ) 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 I f during the cardiac cycle in mouse SAMs. We found that I f 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, I f 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 I f , consistent with a contribution of I f to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously expressed HCN4 channels and by mathematical models of I f Modeling further suggested that the slow rates of activation and deactivation of the HCN4 isoform underlie the persistent activity of I f during the sinoatrial AP. These results establish a new conceptual framework for the role of I f 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., Competing Interests: The authors declare no competing interest.

Properties of the funny current (I f ) have been studied in several animal and cellular models, but so far little is known concerning its properties in human pacemaker cells. This work provides a detailed characterization of I f in human-induced pluripotent stem cell (iPSC)-derived pacemaker cardiomyocytes (pCMs), at different time points. Patch-clamp analysis showed that I f density did not change during differentiation; however, after day 30, it activates at more negative potential and with slower time constants. These changes are accompanied by a slowing in beating rate. I f displayed the voltage-dependent block by caesium and reversed (E rev ) at - 22 mV, compatibly with the 3:1 K + /Na + permeability ratio. Lowering [Na + ] o (30 mM) shifted the E rev to - 39 mV without affecting conductance. Increasing [K + ] o (30 mM) shifted the E rev to - 15 mV with a fourfold increase in conductance. pCMs express mainly HCN4 and HCN1 together with the accessory subunits CAV3, KCR1, MiRP1, and SAP97 that contribute to the context-dependence of I f . Autonomic agonists modulated the diastolic depolarization, and thus rate, of pCMs. The adrenergic agonist isoproterenol induced rate acceleration and a positive shift of I f voltage-dependence (EC 50 73.4 nM). The muscarinic agonists had opposite effects (Carbachol EC 50 , 11,6 nM). Carbachol effect was however small but it could be increased by pre-stimulation with isoproterenol, indicating low cAMP levels in pCMs. In conclusion, we demonstrated that pCMs display an I f with the physiological properties expected by pacemaker cells and may thus represent a suitable model for studying human I f -related sinus arrhythmias.

Knowledge of the histologic constituency of the sinoatrial (SA) node is based on small studies with unevenly distributed ages and subjective assessments of nodal composition, leading to difficulties in interpreting what constitutes true pathology of the SA node. SA nodes from two-hundred normal hearts (10 male and 10 female from each of the first 10 decades of life) were digitally analyzed to assess their histologic composition. Both nodal area and nodal fat content (≥5%) showed a quadratic relationship with age, peaking in the fifth to eighth decades of life. Increased fat content was also more prevalent with increased BMI (≥25 kg/m 2 ). No differences between sexes were observed. Mean nodal collagen ranged from 7.1% to 50.3%, without a statistically significant differences by age or body mass index (BMI). The data suggests that the designation of pathologic fibrosis should be reserved for SA nodes with >50% collagen content. These findings expand and refine our understanding of the anatomy of the SA node., Competing Interests: Declaration of competing interest The authors have no conflicts of interest or funding to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Electrical activity in the heart exhibits 24-hour rhythmicity, and potentially fatal arrhythmias are more likely to occur at specific times of day. Here, we demonstrate that circadian clocks within the brain and heart set daily rhythms in sinoatrial (SA) and atrioventricular (AV) node activity, and impose a time-of-day dependent susceptibility to ventricular arrhythmia. Critically, the balance of circadian inputs from the autonomic nervous system and cardiomyocyte clock to the SA and AV nodes differ, and this renders the cardiac conduction system sensitive to decoupling during abrupt shifts in behavioural routine and sleep-wake timing. Our findings reveal a functional segregation of circadian control across the heart's conduction system and inherent susceptibility to arrhythmia.

[Figure: see text].

Current advanced imaging modalities with applied tracing and processing techniques provide excellent visualization of almost all human internal structures in situ; however, the actual 3D internal arrangement of the human cardiac conduction system (CCS) is still unknown. This study is the first to document the successful 3D visualization of the CCS from the sinus node to the bundle branches within the human body, based on our specialized physical micro-dissection and its CT imaging. The 3D CCS transformation by cardiac inclination changes from the standing to the lying position is also provided. Both actual dissection and its CT image-based simulation identified that when the cardiac inclination changed from standing to lying, the sinus node shifted from the dorso-superior to the right outer position and the atrioventricular conduction axis changed from a vertical to a leftward horizontal position. In situ localization of the human CCS provides accurate anatomical localization with morphometric data, and it indicates the useful correlation between heart inclination and CCS rotation axes for predicting the variable and invisible human CCS in the living body. Advances in future imaging modalities and methodology are essential for further accurate in situ 3D CCS visualization.

Background: It is known that autonomic nerve activity controls the sinus rate. However, the coupling between local nerve activity and electrical activation at the sinoatrial node (SAN) remains unclear. We hypothesized that we would be able to record nerve activity at the SAN to investigate if right stellate ganglion (RSG) activation can increase the local intrinsic nerve activity, accelerate sinus rate, and change the earliest activation sites., Methods: High-density mapping of the epicardial surface of the right atrium including the SAN was performed in 6 dogs during stimulation of the RSG and after RSG stellectomy. A radio transmitter was implanted into 3 additional dogs to record RSG and local nerve activity at the SAN., Results: Heart rate accelerated from 108±4 bpm at baseline to 125±7 bpm after RSG stimulation ( P =0.001), and to 132±7 bpm after apamin injection ( P <0.001). Both electrical RSG stimulation and apamin injection induced local nerve activity at the SAN with the average amplitudes of 3.60±0.72 and 3.86±0.56 μV, respectively. RSG stellectomy eliminated the local nerve activity and decreased the heart rate. In ambulatory dogs, local nerve activity at the SAN had a significantly higher average Pearson correlation to heart rate (0.72±0.02, P =0.001) than RSG nerve activity to HR (0.45±0.04, P =0.001)., Conclusions: Local intrinsic nerve activity can be recorded at the SAN. Short bursts of these local nerve activities are present before each atrial activation during heart rate acceleration induced by stimulation of the RSG.

The sinus node (SAN) is the primary pacemaker of the human heart, and abnormalities in its structure or function cause sick sinus syndrome, the most common reason for electronic pacemaker implantation. Here we report that transcription factor GATA6, whose mutations in humans are linked to arrhythmia, is highly expressed in the SAN and its haploinsufficiency in mice results in hypoplastic SANs and rhythm abnormalities. Cell-specific deletion reveals a requirement for GATA6 in various SAN lineages. Mechanistically, GATA6 directly activates key regulators of the SAN genetic program in conduction and nonconduction cells, such as TBX3 and EDN1, respectively. The data identify GATA6 as an important regulator of the SAN and provide a molecular basis for understanding the conduction abnormalities associated with GATA6 mutations in humans. They also suggest that GATA6 may be a potential modifier of the cardiac pacemaker., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Na ionophores increase intracellular Na + ([Na + ]i). Membrane potentials and currents were measured using microelectrode and whole-cell patch-clamp techniques. Monensin (10 -6 -3×10 -5 M) reduced the slope of the pacemaker potentials and shortened the action potential duration (APD) in sino-atrial nodal and Purkinje cells. Monensin (10 -5 M) shortened the APD and reduced the amplitude of the plateau phase in ventricular myocytes. Monensin decreased the hyperpolarization-activated inward current (I f ), and it increased the transient outward potassium current (I to ) in Purkinje cells. In addition, monensin decreased the sodium current (I Na ), shifting the inactivation curve to the hyperpolarized direction. Moreover, monensin decreased the L-type calcium current (I Ca ) in ventricular myocytes. The Na + -Ca 2+ exchange current (I Na-Ca ) was augmented particularly in the reverse mode, and the Na + -K + pump current (I Na-K ) was also activated by monensin in cardiomyocytes. The ATP-activated potassium current (I K,ATP ) could be induced by monensin. Notably, the inward rectifying K + current (I K1 ), and the slow delayed outward K + current (I Ks ) were not affected evidently by monensin. Collectively, alteration of [Na + ]i can influence the activities of various ion channels and transporters. Thus, the significance of altered [Na + ]i should be taken into consideration in the action of drugs affecting [Na + ]i such as digitalis, Na + channel blockers, and Na + channel activating agents.

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.)

Background The sinus node (SN) is the primary pacemaker of the heart. SN myocytes possess distinctive action potential morphology with spontaneous diastolic depolarization because of a unique expression of ion channels and Ca 2+ -handling proteins. MicroRNAs (miRs) inhibit gene expression. The role of miRs in controlling the expression of genes responsible for human SN pacemaking and conduction has not been explored. The aim of this study was to determine miR expression profile of the human SN as compared with that of non-pacemaker atrial muscle. Methods and Results SN and atrial muscle biopsies were obtained from donor or post-mortem hearts (n=10), histology/immunolabeling were used to characterize the tissues, TaqMan Human MicroRNA Arrays were used to measure 754 miRs, Ingenuity Pathway Analysis was used to identify miRs controlling SN pacemaker gene expression. Eighteen miRs were significantly more and 48 significantly less abundant in the SN than atrial muscle. The most interesting miR was miR-486-3p predicted to inhibit expression of pacemaking channels: HCN1 (hyperpolarization-activated cyclic nucleotide-gated 1), HCN4, voltage-gated calcium channel (Ca v )1.3, and Ca v 3.1. A luciferase reporter gene assay confirmed that miR-486-3p can control HCN4 expression via its 3' untranslated region. In ex vivo SN preparations, transfection with miR-486-3p reduced the beating rate by ≈35±5% ( P <0.05) and HCN4 expression ( P <0.05). Conclusions The human SN possesses a unique pattern of expression of miRs predicted to target functionally important genes. miR-486-3p has an important role in SN pacemaker activity by targeting HCN4, making it a potential target for therapeutic treatment of SN disease such as sinus tachycardia.

Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary proteins. Here, we report the discovery of a family of endoplasmic reticulum (ER) transmembrane proteins that associate with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, and Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. Despite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depolarizing shift in the voltage dependence of HCN4 in response to the binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage dependence in the absence of cAMP. The mechanisms of action of LRMP and IRAG are independent of trafficking and cAMP binding, and they are specific to the HCN4 isoform. We also found that IRAG is highly expressed in the mouse sinoatrial node where computer modeling predicts that its presence increases HCN4 current. Our results suggest important roles for LRMP and IRAG in the regulation of cellular excitability, as tools for advancing mechanistic understanding of HCN4 channel function, and as possible scaffolds for coordination of signaling pathways., Competing Interests: The authors declare no competing interest.

Heart rate and heart rate variability (HRV) are mainly determined by the autonomic nervous system (ANS), which interacts with receptors on the sinoatrial node (SAN; the heart's primary pacemaker), and by the "coupled-clock" system within the SAN cells. HRV changes are associated with cardiac diseases. However, the relative contributions of the ANS and SAN to HRV are not clear, impeding effective treatment. To discern the SAN's contribution, we performed HRV analysis on canine electrocardiograms containing basal and ANS-blockade segments. We also analyzed human electrocardiograms of atrial fibrillation and heart failure patients, as well as healthy aged subjects. Finally, we used a mathematical model to simulate HRV under decreased "coupled-clock" regulation. We found that (a) in canines, the SAN and ANS contribute mainly to long- and short-term HRV, respectively; (b) there is evidence suggesting a similar relative SAN contribution in humans; (c) SAN features can be calculated from beat-intervals obtained in-vivo, without intervention; (d) ANS contribution can be modeled by sines embedded in white noise; (e) HRV changes associated with cardiac diseases and aging can be interpreted as deterioration of both SAN and ANS; and (f) SAN clock-coupling can be estimated from changes in HRV. This may enable future non-invasive diagnostic applications.

Rationale: The stress response of heart rate, which is determined by the plasticity of the sinoatrial node (SAN), is essential for cardiac function and survival in mammals. As an RNA-binding protein, CIRP (cold-inducible RNA-binding protein) can act as a stress regulator. Previously, we have documented that CIRP regulates cardiac electrophysiology at posttranscriptional level, suggesting its role in SAN plasticity, especially upon stress conditions., Objective: Our aim was to clarify the role of CIRP in SAN plasticity and heart rate regulation under stress conditions., Methods and Results: Telemetric ECG monitoring demonstrated an excessive acceleration of heart rate under isoprenaline stimulation in conscious CIRP-KO (knockout) rats. Patch-clamp analysis and confocal microscopic Ca 2+ imaging of isolated SAN cells demonstrated that isoprenaline stimulation induced a faster spontaneous firing rate in CIRP-KO SAN cells than that in WT (wild type) SAN cells. A higher concentration of cAMP-the key mediator of pacemaker activity-was detected in CIRP-KO SAN tissues than in WT SAN tissues. RNA sequencing and quantitative real-time polymerase chain reaction analyses of single cells revealed that the 4B and 4D subtypes of PDE (phosphodiesterase), which controls cAMP degradation, were significantly decreased in CIRP-KO SAN cells. A PDE4 inhibitor (rolipram) abolished the difference in beating rate resulting from CIRP deficiency. The mechanistic study showed that CIRP stabilized the mRNA of Pde4b and Pde4d by direct mRNA binding, thereby regulating the protein expression of PDE4B and PDE4D at posttranscriptional level., Conclusions: CIRP acts as an mRNA stabilizer of specific PDEs to control the cAMP concentration in SAN, maintaining the appropriate heart rate stress response.

Background: Atrial fibrillation (AF) is the most frequent cardiac arrhythmia which increases the risk of thromboembolic complications and impairs quality of life. An important part of a therapeutic approach for AF is sinus rhythm restoration. Antiarrhythmic agents used in pharmacological cardioversion have limited efficacy and potential risk of proarrhythmia. Simultaneously, underlying conditions of AF should be treated (e.g. electrolyte imbalance, increased blood pressure, neurohormonal disturbances, atrial volume overload). There is still the need for an effective and safe approach to increase AF cardioversion efficacy. This randomized, double-blind, placebo-controlled, superiority clinical study is performed in patients with AF in order to evaluate the clinical efficacy of intravenous canrenone in sinus rhythm restoration., Methods: Eighty eligible patients with an episode of AF lasting less than 48 h are randomized in a 1:1 ratio to receive canrenone or placebo. Patients randomized to a treatment intervention are receiving canrenone intravenously at a dose of 200 mg within 2-3 min. Subjects assigned to a control group obtain the same volume of 0.9% saline within the same time. The primary endpoint includes return of sinus rhythm documented in the electrocardiogram within 2 h after drug or placebo administration. Other endpoints and safety outcomes analyses, due to expected lack of statistical power, are exploratory., Discussion: Current evidence supports renin-angiotensin-aldosterone system (RAAS) inhibition as an upstream therapy in AF management. Excess aldosterone secretion results in proarrhythmic effects. Among the RAAS inhibitors, only canrenone is administered intravenously. Canrenone additionally increases the plasma level of potassium, lowers blood pressure and reduces preload. It has been already used in primary and secondary hyperaldosteronism in the course of chronic liver dysfunction and in heart failure., Trial Registration: ClinicalTrials.gov, NCT03536806. Registered on 25 May 2018.

A progressive decline in maximum heart rate (mHR) is a fundamental aspect of aging in humans and other mammals. This decrease in mHR is independent of gender, fitness, and lifestyle, affecting in equal measure women and men, athletes and couch potatoes, spinach eaters and fast food enthusiasts. Importantly, the decline in mHR is the major determinant of the age-dependent decline in aerobic capacity that ultimately limits functional independence for many older individuals. The gradual reduction in mHR with age reflects a slowing of the intrinsic pacemaker activity of the sinoatrial node of the heart, which results from electrical remodeling of individual pacemaker cells along with structural remodeling and a blunted β-adrenergic response. In this review, we summarize current evidence about the tissue, cellular, and molecular mechanisms that underlie the reduction in pacemaker activity with age and highlight key areas for future work.

Sinus node dysfunction (SND) is often associated with atrial fibrillation (AF). Amiodarone is the most frequently used agent for maintaining sinus rhythm in patients with AF, but it impairs the sinoatrial node (SAN) function in one-third of AF patients. This study aims to gain mechanistic insights into the effects of the antiarrhythmic agents in the setting of AF-induced SND. We have adapted a human SAN model to characterize the SND conditions by incorporating experimental data on AF-induced electrical remodelling, and then integrated actions of drugs into the modified model to assess their efficacy. Reductions in pacing rate upon the implementation of AF-induced electrical remodelling associated with SND agreed with the clinical observations. And the simulated results showed the reduced funny current (I f ) in these remodelled targets mainly contributed to the heart rate reduction. Computational drug treatment simulations predicted a further reduction in heart rate during amiodarone administration, indicating that the reduction was the result of actions of amiodarone on I Na , I Kur , I CaL , I CaT , I f and beta-adrenergic receptors. However, the heart rate was increased in the presence of disopyramide. We concluded that disopyramide may be a desirable choice in reversing the AF-induced SND phenotype.

This study investigated whether overexpression of paired-related homeobox 1 (prrx1) can successfully induce differentiation of brown adipose-derived stem cells (BADSCs) into sinus node-like cells. The experiments were performed in two groups: adenovirus-green fluorescent protein (Ad-GFP) group and Ad-prrx1 group. After 5-7 days of adenoviral transfection, the expression levels of sinus node cell-associated pacing protein (hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 [HCN4]) and ion channel (calcium channel, voltage-dependent, T type, alpha 1G subunit [Cacna1g]), as well as transcription factors (T-box 18 [TBX18], insulin gene enhancer binding protein 1 [ISL-1], paired-like homeodomain transcription factor 2 [pitx2], short stature homeobox 2 [shox2]), were detected by western blot and reverse transcription-quantitative polymerase chain reaction. Immunofluorescence assay was carried out to detect whether prrx1 was coexpressed with HCN4, TBX18, and ISL-1. Finally, whole-cell patch-clamp technique was used to record pacing current hyperpolarization-activated inward current (I f ). The isolated cells were CD90 + , CD29 + , and CD45 - , indicating that pure BADSCs were successfully isolated. After 5-7 days of Ad transfection into cells, the mRNA levels and protein levels of pacing-related factors (TBX18, ISL-1, HCN4, shox2, and Cacna1g) in Ad-prrx1 group were significantly higher than those in Ad-GFP group. However, the expression level of pitx2 was decreased. Immunofluorescence analysis showed that prrx1 was coexpressed with TBX18, ISL-1, and HCN4 in the Ad-prrx1 group, which did not appear in the Ad-GFP group. Whole-cell patch clamps were able to record the I f current in the experimental group rather than in the Ad-GFP group. Overexpression of prrx1 can successfully induce sinus node-like cells.

The cardiac nodes are the source of the electrical impulse that is transmitted to the heart, the aim of this work is study the histological and morphometric characteristics of the different components of the sinus and atrioventricular nodes in horses and dogs that help to know the physiopathology of these nodes. A group of ten horse hearts and five dog hearts were used. The region of the sinus and atrioventricular nodes was sectioned serially, and the block of tissue removed for study. The samples were assessed using a morphometric analysis with the Image-Pro Plus 7.1 software and the acquisition of the images using a Leica DMD108 optic microscope. The shape of the horse's sinus node is oblong and its P cells are large. The shape of the dog's sinus is rounded or oblong. The P cells are large and pale. The area of P cells in horses was 976 (SD 223.7) μm 2 and in dogs the area for P cells was 106 (SD 30.4) μm 2 , which indicates that the value for P cells in horses are significantly higher than in dogs (p = .001). The horse atrioventricular node presented an oblong shape and in dogs, presents a spindle shape. The lower cell density in any of the cardiac nodes, especially in P cells of sinus node, can decrease electrical conduction within the nodes and in the internodal tracts, which would reflect the presence of cardiac arrhythmias derived from poor conduction, even in morphologically normal hearts., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Heart rate in physiological conditions is set by the sinoatrial node (SN), the primary cardiac pacing tissue. Phosphoinositide 3-kinase (PI3K) signaling is a major regulatory pathway in all normal cells, and its dysregulation is prominent in diabetes, cancer, and heart failure. Here, we show that inhibition of PI3K slows the pacing rate of the SN in situ and in vitro and reduces the early slope of diastolic depolarization. Furthermore, inhibition of PI3K causes a negative shift in the voltage dependence of activation of the pacemaker current, I F , while addition of its second messenger, phosphatidylinositol 3,4,5-trisphosphate, induces a positive shift. These shifts in the activation of I F are independent of, and larger than, those induced by the autonomic nervous system. These results suggest that PI3K is an important regulator of heart rate, and perturbations in this signaling pathway may contribute to the development of arrhythmias., (© 2019 Lin et al.)

The sinoatrial node (SAN), the primary cardiac pacemaker, consists of a head domain and a junction/tail domain that exhibit different functional properties. However, the underlying molecular mechanism defining these two pacemaker domains remains elusive. Nkx2-5 is a key transcription factor essential for the formation of the working myocardium, but it was generally thought to be detrimental to SAN development. However, Nkx2-5 is expressed in the developing SAN junction, suggesting a role for Nkx2-5 in SAN junction development and function. In this study, we present unambiguous evidence that SAN junction cells exhibit unique action potential configurations intermediate to those manifested by the SAN head and the surrounding atrial cells, suggesting a specific role for the junction cells in impulse generation and in SAN-atrial exit conduction. Single-cell RNA-seq analyses support this concept. Although Nkx2-5 inactivation in the SAN junction did not cause a malformed SAN at birth, the mutant mice manifested sinus node dysfunction. Thus, Nkx2-5 defines a population of pacemaker cells in the transitional zone. Despite Nkx2-5 being dispensable for SAN morphogenesis during embryogenesis, its deletion hampers atrial activation by the pacemaker., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Key Points: Vagal reflexes slow heart rate and can change where the heartbeat originates within the sinoatrial node (SAN). The mechanisms responsible for this process - termed leading pacemaker (LP) shift - have not been investigated fully. We used optical mapping to measure the effects of baroreflex, chemoreflex and carbachol on pacemaker entrainment and electrical conduction across the SAN. All methods of stimulation triggered shifts in LP site from the central SAN to one or two caudal pacemaker regions. These shifts were associated with reduced current generation capacity centrally and increased electrical load caudally. Previous studies suggest LP shift is a rate-dependent phenomenon whereby acetylcholine slows central pacemaker rate disproportionately, enabling caudal cells that are less acetylcholine sensitive to assume control. However, our findings indicate the LP region is defined by both pacemaker rate and capacity to drive activation. Shifts in LP site provide an important homeostatic mechanism for rapid switches in heart rate., Abstract: Reflex vagal activity causes abrupt heart rate slowing with concomitant caudal shifts of the leading pacemaker (LP) site within the sinoatrial node (SAN). However, neither the mechanisms responsible nor their dynamics have been investigated fully. Therefore, the objective of this study was to elucidate the mechanisms driving cholinergic LP shift. Optical maps of right atrial activation were acquired in a rat working heart-brainstem preparation during baroreflex and chemoreflex stimulation or with carbachol. All methods of stimulation triggered shifts in LP site from the central SAN to caudal pacemaker regions, which were positive for HCN4 and received uniform cholinergic innervation. During baroreflex onset, the capacity of the central region to drive activation declined with a decrease in amplitude and gradient of optical action potentials (OAPs) in the surrounding myocardium. Accompanying this decline, there was altered entrainment in the caudal SAN as shown by decreased conduction velocity, OAP amplitude, gradient and activation time. Atropine abolished these responses. Chemoreflex stimulation produced similar effects but central capacity to drive activation was preserved before the LP shift. In contrast, carbachol produced a prolonged period of reduced capacity to drive and altered entrainment. Previous studies suggest LP shift is a rate-dependent phenomenon whereby acetylcholine slows central pacemaker rate disproportionately, enabling caudal cells that are less acetylcholine sensitive to assume control. Our findings indicate that cholinergic LP shifts are also determined by altered electrical source-to-sink balance in the SAN. We conclude that the LP region is defined by both rate and capacity to drive atrial activation., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

We hypothesized that the repolarization phase of action potentials (APs) in mammals with large body mass and high cardiac output could not be reliably controlled by only one of the delayed rectifier potassium I K current components. To test this hypothesis experimentally, we performed a comparative study of the response of AP phases to the rapid I Kr channels blocker E-4031 and slow I Ks blocker chromanol 293B in APs spontaneously generated in strips of sinoauricular (SA) tissue from mouse, guinea pig, and pig hearts. Application of a slow channels blocker chromanol 293B caused a decrease of Aps generation frequency in SA area strips from mouse, guinea-pig and pig by 5.3, 16, and 18% compared to the control. Treatment with the I Kr blocker E-4031 caused a significant reduction of APs generation frequency in the mouse, guinea pig, and pig SA strips by 24, 26, and 36%, respectively, compared to the control values. These results suggest that the rapid I Kr current is the key component responsible for AP generation in sinoauricular node cells of the pig heart.