Human Sinoatrial Node Research Articles

Reproducibility study of the Fabbri et al. 2017 model of the human sinus node action potential

The sinoatrial node (SAN) is the natural pacemaker of the mammalian heart. It has been the subject of several mathematical studies, aimed at reproducing its electrical response under normal sinus rhythms, as well as under various conditions. Such studies were traditionally done using data from rabbit SAN cells. More recently, human SAN cell data have become available, resulting in the publication of a human SAN cell model (Fabbri et al., 2017), along with its CellML version. Here, we used the CellML file provided by the model authors, together with some SED-ML files and Python scripts that we created to reproduce the main results of the aforementioned modeling study. EDITOR'S NOTE (v2): this article and its OMEX archive are republished with technical changes made to the corresponding Python scripts to remove a run-time error message displayed when executing each simulation.

Reproducibility study of the Fabbri et al. 2017 model of the human sinus node action potential

The sinoatrial node (SAN) is the natural pacemaker of the mammalian heart. It has been the the subject of several mathematical studies, aimed at reproducing its electrical response under normal sinus rhythms, as well as under various conditions. Such studies were traditionally done using data from rabbit SAN cells. More recently, human SAN cell data have become available, resulting in the publication of a human SAN cell model (Fabbri et al., 2017), along with its CellML version. Here, we used the CellML file provided by the model authors, together with some SED-ML files and Python scripts that we created to reproduce the main results of the aforementioned modeling study.

The Functional Role of Hyperpolarization Activated Current (I f) on Cardiac Pacemaking in Human vs. in the Rabbit Sinoatrial Node: A Simulation and Theoretical Study.

The cardiac hyperpolarization-activated “funny” current (If), which contributes to sinoatrial node (SAN) pacemaking, has a more negative half-maximal activation voltage and smaller fully-activated macroscopic conductance in human than in rabbit SAN cells. The consequences of these differences for the relative roles of If in the two species, and for their responses to the specific bradycardic agent ivabradine at clinical doses have not been systematically explored. This study aims to address these issues, through incorporating rabbit and human If formulations developed by Fabbri et al. into the Severi et al. model of rabbit SAN cells. A theory was developed to correlate the effect of If reduction with the total inward depolarising current (Itotal) during diastolic depolarization. Replacing the rabbit If formulation with the human one increased the pacemaking cycle length (CL) from 355 to 1,139 ms. With up to 20% If reduction (a level close to the inhibition of If by ivabradine at clinical concentrations), a modest increase (~5%) in the pacemaking CL was observed with the rabbit If formulation; however, the effect was doubled (~12.4%) for the human If formulation, even though the latter has smaller If density. When the action of acetylcholine (ACh, 0.1 nM) was considered, a 20% If reduction markedly increased the pacemaking CL by 37.5% (~27.3% reduction in the pacing rate), which is similar to the ivabradine effect at clinical concentrations. Theoretical analysis showed that the resultant increase of the pacemaking CL is inversely proportional to the magnitude of Itotal during diastolic depolarization phase: a smaller If in the model resulted in a smaller Itotal amplitude, resulting in a slower pacemaking rate; and the same reduction in If resulted in a more significant change of CL in the cell model with a smaller Itotal. This explained the mechanism by which a low dose of ivabradine slows pacemaking rate more in humans than in the rabbit. Similar results were seen in the Fabbri et al. model of human SAN cells, suggesting our observations are model-independent. Collectively, the results of study explain why low dose ivabradine at clinically relevant concentrations acts as an effective bradycardic agent in modulating human SAN pacemaking.

HCN4 current during human sinoatrial node-like action potentials

BackgroundDespite the many studies carried out over the past 40 years, the contribution of the HCN4 encoded hyperpolarization-activated ‘funny’ current (If) to pacemaker activity in the mammalian sinoatrial node (SAN), and the human SAN in particular, is still controversial and not fully established. ObjectiveTo study the contribution of If to diastolic depolarization of human SAN cells and its dependence on heart rate, cAMP levels, and atrial load. MethodsHCN4 channels were expressed in human cardiac myocyte progenitor cells (CMPCs) and HCN4 currents assessed using perforated patch-clamp in traditional voltage clamp mode and during action potential clamp with human SAN-like action potential waveforms with 500–1500 ms cycle length, in absence or presence of forskolin to mimic β-adrenergic stimulation and a −15 mV command potential offset to mimic atrial load. ResultsForskolin significantly increased the fully-activated HCN4 current density at −140 mV by 14% and shifted the steady-state activation curve by +7.4 mV without affecting its slope. In addition, forskolin significantly accelerated current activation but slowed deactivation. The HCN4 current did not completely deactivate before the subsequent diastolic depolarization during action potential clamp. The amplitude of HCN4 current increased with increasing cycle length, was significantly larger in the presence of forskolin at all cycle lengths, and was significantly increased upon the negative offset to the command potential. ConclusionsIf is active during a human SAN action potential waveform and its amplitude is modulated by heart rate, β-adrenergic stimulation, and diastolic voltage range, such that If is under delicate control.

Do human sinoatrial node cells have t-tubules?

Every heartbeat is generated by spontaneously depolarizing specialised nodal cells that make up the sinoatrial node (SN). The SN's unique properties depend on two complex mechanisms - the membrane-voltage and the Ca2+ clocks, regulated by strictly defined expression of ion channels, connexins and Ca2+-handling proteins. In the working myocardium these proteins are accumulated in specialised membrane structures– the transverse tubules (t-tubules). It is well known that small mammals do not exhibit t-tubules in their atrium, but such structures are observed in the atria of bigger mammals such as sheep. No information on t-tubules in the human SN is present yet. The aim of our study was to determine if t-tubules are present in the human SN, as well as surrounding atrial myocardium. Dystrophin (cytoplasmic protein connecting the muscle fibres to the surrounding extracellular membrane), Na+-Ca2+ exchanger (NCX1), sarcoplasmic-endoplasmic reticulum calcium adenosine triphosphatase (SERCA2a), α-actinin, and ryanodine receptor 2 (RyR2) were investigated. Staining with wheat germ agglutinin (WGA, specific marker of t-tubules) demonstrated that human RA cells contain t-tubules, but these structures are very sparse in the SN. Experiments in single nodal cells from rat and mouse showed no expression of t-tubular network, but striated pattern of expression of RyR2 was obtained similar to the human sections, suggesting its manner of organization is dependent on α-actinin colocalization.

Speeding Up the Heart? Traditional and New Perspectives on HCN4 Function.

The sinoatrial node (SAN) is the primary pacemaker of the heart and is responsible for generating the intrinsic heartbeat. Within the SAN, spontaneously active pacemaker cells initiate the electrical activity that causes the contraction of all cardiomyocytes. The firing rate of pacemaker cells depends on the slow diastolic depolarization (SDD) and determines the intrinsic heart rate (HR). To adapt cardiac output to varying physical demands, HR is regulated by the autonomic nervous system (ANS). The sympathetic and parasympathetic branches of the ANS innervate the SAN and regulate the firing rate of pacemaker cells by accelerating or decelerating SDD–a process well-known as the chronotropic effect. Although this process is of fundamental physiological relevance, it is still incompletely understood how it is mediated at the subcellular level. Over the past 20 years, most of the work to resolve the underlying cellular mechanisms has made use of genetically engineered mouse models. In this review, we focus on the findings from these mouse studies regarding the cellular mechanisms involved in the generation and regulation of the heartbeat, with particular focus on the highly debated role of the hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 in mediating the chronotropic effect. By focusing on experimental data obtained in mice and humans, but not in other species, we outline how findings obtained in mice relate to human physiology and pathophysiology and provide specific information on how dysfunction or loss of HCN4 channels leads to human SAN disease.

Fibroblast-Specific Proteotranscriptomes Reveal Distinct Fibrotic Signatures of Human Sinoatrial Node in Nonfailing and Failing Hearts.

Up to 50% of the adult human sinoatrial node (SAN) is composed of dense connective tissue. Cardiac diseases including heart failure (HF) may increase fibrosis within the SAN pacemaker complex, leading to impaired automaticity and conduction of electric activity to the atria. Unlike the role of cardiac fibroblasts in pathologic fibrotic remodeling and tissue repair, nothing is known about fibroblasts that maintain the inherently fibrotic SAN environment. Intact SAN pacemaker complex was dissected from cardioplegically arrested explanted nonfailing hearts (non-HF; n=22; 48.7±3.1 years of age) and human failing hearts (n=16; 54.9±2.6 years of age). Connective tissue content was quantified from Masson trichrome-stained head-center and center-tail SAN sections. Expression of extracellular matrix proteins, including collagens 1 and 3A1, CILP1 (cartilage intermediate layer protein 1), and POSTN (periostin), and fibroblast and myofibroblast numbers were quantified by in situ and in vitro immunolabeling. Fibroblasts from the central intramural SAN pacemaker compartment (≈10×5×2 mm3) and right atria were isolated, cultured, passaged once, and treated ± transforming growth factor β1 and subjected to comprehensive high-throughput next-generation sequencing of whole transcriptome, microRNA, and proteomic analyses. Intranodal fibrotic content was significantly higher in SAN pacemaker complex from HF versus non-HF hearts (57.7±2.6% versus 44.0±1.2%; P<0.0001). Proliferating phosphorylated histone 3+/vimentin+/CD31- (cluster of differentiation 31) fibroblasts were higher in HF SAN. Vimentin+/α-smooth muscle actin+/CD31- myofibroblasts along with increased interstitial POSTN expression were found only in HF SAN. RNA sequencing and proteomic analyses identified unique differences in mRNA, long noncoding RNA, microRNA, and proteomic profiles between non-HF and HF SAN and right atria fibroblasts and transforming growth factor β1-induced myofibroblasts. Specifically, proteins and signaling pathways associated with extracellular matrix flexibility, stiffness, focal adhesion, and metabolism were altered in HF SAN fibroblasts compared with non-HF SAN. This study revealed increased SAN-specific fibrosis with presence of myofibroblasts, CILP1, and POSTN-positive interstitial fibrosis only in HF versus non-HF human hearts. Comprehensive proteotranscriptomic profiles of SAN fibroblasts identified upregulation of genes and proteins promoting stiffer SAN extracellular matrix in HF hearts. Fibroblast-specific profiles generated by our proteotranscriptomic analyses of the human SAN provide a comprehensive framework for future studies to investigate the role of SAN-specific fibrosis in cardiac rhythm regulation and arrhythmias.

Genetic Complexity of Sinoatrial Node Dysfunction.

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.

Endo-Epicardial Mapping of InVivo Human Sinoatrial Node Activity.

The aim of the current study was to examine electrophysiological characteristics of sinoatrial node (SAN) activity from an endo-epicardial perspective. Electrophysiological properties of the invivo human SAN and its exit pathways remain poorly understood. Twenty patients (75% male; median age 66 years [59 to 73 years]) with structural heart disease underwent simultaneous endo-epicardial mapping (256 unipolar electrodes, interelectrode distance 2mm). Conduction times, endo-epicardial delays (EEDs), and R/S ratio were examined in the surrounding 10mm of SAN activation. Areas of conduction block were defined as conduction delays≥12ms and endo-epicardial asynchrony as EED≥15 m. Three distinct activation patterns were observed in a total of 28 SAN-focal activation patterns (SAN-FAPs) (4 patients exhibited >1 different exit site), including SAN activation patterns with: 1) solely an endocardial exit site (n=10 [36%]); 2) solely an epicardial exit site (n=13 [46%]); and 3) simultaneously activated endo-epicardial exit sites (n=5 [18%]). Median (interquartile range) EED at the origin of the SAN-FAP was 10ms (6 to 14 ms) and the prevalence of endo-epicardial asynchrony in the surroundings of the SAN-FAP was 5% (2% to 18%). Electrograms at the origin of the SAN-FAPs exhibited significantly larger R-peaks in the mid right atrium (RA) compared with the superior RA (mid R/S ratio 0.15 [0.067 to 0.34] vs. superior R/S ratio 0.045 [0.026 to 0.062]; p=0.004). Conduction velocity within a distance of 10mm from the SAN-FAP was 125cm/s (80 to 250 cm/s). All 6 SAN-FAPs at the mid RA were observed in patients with a history of atrial fibrillation. Variations in activation patterns of the SAN observed in this study highlight the complex 3-dimensional SAN geometry and indicate the presence of interindividual differences in SAN exit pathways. Solely in patients with a history of atrial fibrillation, SAN activity occurred more caudally, which indicates changes in preferential SAN exitpathways.

Modeling the Human Sinoatrial Node Based on Sequential Discharge Hypothesis

The electrical synchronization of human sinoatrial (SA) node cells for propagating the pacemaker activity has always been an important challenge in literatures. Nevertheless, most literatures, that provide hypotheses for the electrical synchronization of SA node cells, only investigated the activity of a very small group of coupled SA node cells. Therefore, this study proposes a novel SA node model based on the sequential discharge hypothesis to estimate the electrical potential around the SA node, which electrodes measure it. This model consists of a three-dimensional regular network of Hodgkin-Huxley (HH) model modified by time constantthat propagates the action potentials of dominant pacemaker through the gap junctions. The results show that the HH model modified in this study not only has adequate capacity for generating oscillations between 40 and 180 beats per minute (BPM), but also their three-dimensional regular network can provide a suitable estimate from the electrical potential around the SA node. Therefore, this model is a good suggestion for the electrical synchronization of large mammalian SA node cells, especially human.

Electrophysiological characteristics of telocytes of the atrioventricular note and sinoatrial node perifocal area in humans and pigs

Little is known about the electrophysiological characteristics of telocytes found in the working myocardium and sinoatrial node (SAN). Telocyte expres -sion of HCN4 suggests the ability to generate pacemaker potentials. To prove the impulse conduction, the presence of voltage-gated sodium channels is required. It is assumed that telocytes are also located in the atrioventricular node (AVN). Aim . Morphological and electrophysiological study of AVN and SAN telocytes. Material and methods. Fragments of the right atrium, AVN of 7 hearts of recipients and 3 hearts of pigs were taken, respectively, during heart transplantation and after the experiment. Isolation of telocyte cultures, histological, immunohis tochemical tests with anti-CD117, anti-NaV1.5 (SCN5A), anti-CD34 antibodies, intravital confocal laser microscopy were carried out. The patch-clamp technique was used. Results . In AVN cultures, CD117 + cells with long processes were found. There was a potassium current with a density of 700 pA/pF during membrane depolarization up to +90 mW in humans and pigs using the patch-clamp technique. Calcium oscillations with a period of about 200 seconds in a pig with an increase in calcium concentration. In elongated cells located between cardiomyocytes and among the fibrous tissue of SAN perifocal zone, the co-expression of anti-NaV1.5 and anti-CD34 antibodies was revealed. Conclusion . In AVN, telocytes were found, in whose cultures potassium current and calcium oscillations were determined. SCN5A sodium channels were found in telocytes of the perifocal area of human SAN. This fact indicates the ability of cells to conduct an electrical impulse.

Abstract 14223: Identifying Pro-fibrotic Long Non-coding RNAs Ii Fibroblasts From the Failing and Non-failing Human Sinoatrial Node

Introduction: Dense fibrous connective tissue is inherently found in the human sinoatrial node (hSAN), which further increases in heart failure (HF) leading to sinoatrial node dysfunction (SND). While several factors contribute to cardiac fibrosis, it is unknown if long non-coding RNAs (lncRNA), a novel class of RNA known to affect cardiac fibrosis, are involved in increasing fibrotic content in hSAN in non-failing (nHF) vs HF hearts. Objective: To identify unique lncRNA profiles and pro-fibrotic lncRNAs in isolated SAN and right atrial (RA) fibroblasts (FBs), in HF vs nHF human hearts. Methods: FBs isolated from pure SAN and RA tissues, from HF (n=6; 35-68yo) and nHF (n=4; 26-64yo) cardioplegically arrested human hearts, were cultured with/without transforming growth factor β1 (TGFβ1; 5ng; 48hrs) to activate myofibroblast (myoFB) transition. Frozen FBs and myoFBs were subjected to high throughput Next Generation RNA Sequencing analyses of the whole transcriptome. Results: Averages of total counts across all samples revealed that majority of the genes detected were protein coding 14415(63%), 5876 (26%) lncRNA, 1254 (5%) miscellaneous RNA, 677(3%) miRNA and 577 (3%) other types of RNA (Figure A). Preliminary analyses show that coding mRNA and non-coding lncRNA are differentially expressed in nHF hSAN and RA fibroblasts and TGFβ1 treated myoFBs. Furthermore, these expression patterns were also different in FBs and myoFBs isolated from failing hearts. Conclusions: Our findings show for the first time that lncRNA expression in cultured hSAN Fbs and myoFBs are unique and differentially altered in HF. Ongoing analyses of sequenced transcriptome will identify FB and myoFB lncRNAs associated with intrinsic higher levels of hSAN fibrotic content as well as in HF. We will also determine if they can modify pro-fibrotic activity in HF SAN FBs and myoFBs, which may be beneficial to develop novel molecular approaches to decrease HF-associated SAN fibrosis and associated SND.

Abstract 17006: Reversing Membrane Clock Remodeling (MCR) Associated With Heart Failure With Preserved Ejection Fraction (HFpEF) in the Sinoatrial Node (SAN) Eliminates Chronotropic Incompetence (CI)

Introduction: While CI, or the inability of heart rate (HR) to adequately respond to exercise, is a strong indicator of mortality in HFpEF, treatment options remain limited as its mechanisms remain underexplored. Ionic remodeling of the SAN, both of the membrane (MC) and calcium clocks (CC), is associated with HFpEF and CI. Aim: Using an in silico approach, we investigated whether SAN ionic remodeling can contribute to 3 different CI presentations (Fig A) and explored the effectiveness of targeting the remodeling of the MC or CC (MCR and CCR, resp.) in eliminating CI. Methods: A human SAN model (control) was used, in which ionic remodeling was integrated using experimental data from HFpEF rat model with CI (Fig B top). A family of HFpEF SAN models (n = 147) was generated because the uniqueness of Ca 2+ handling parameters constrained to experimentally-based Ca 2+ transients could not be guaranteed (Fig B middle). To assess the level of CI, max beat rate (BR), BR response (τ on ), and BR recovery (τ off ) were quantified for each model in response to a transient 40s pulse of 1 μM isoproterenol (ISO) (Fig B bottom). Lastly, the effect of reversing either CCR or MCR on CI was determined. Results: Without ISO, BR of HFpEF SAN models was similar to that of control; when ISO was applied (Fig C), HFpEF models exhibited all aspects of CI: submaximal BR (Fig D), and elevated τ on and τ off (Fig E). In CCR reversed models, BR without ISO was similar to that of control; with ISO, they exhibited submaximal BR (Fig F). In MCR reversed models, BR without ISO was slightly lower compared to control; with ISO, while they exhibited submaximal BR, the increase in BR with ISO was similar to that of control (Fig F). τ on and τ off were elevated in CCR reversed models, but they were not in MCR reversed models (Fig G). In summary, CCR reversed models exhibited all aspects of CI, but MCR reversed models did not. Conclusion: Ionic remodeling of the SAN contributes to CI in HFpEF, and targeting MCR could be a promising strategy for eliminating CI.

Simultaneous endo-epicardial mapping of the human sinus node in patients undergoing cardiac surgery

Abstract Introduction Conduction disorders in the right atrium (RA) are predominantly observed in the superior part. Presence of the sinoatrial node (SAN) in this region could be contributing to increased heterogeneity in activation patterns. However, mapping studies focusing on SAN activation are scarce. Purpose The aim of the present study was to analyze activation patterns of the human SAN during sinus rhythm. Methods A dataset containing simultaneous endo-epicardial mapping data (256 electrodes, inter-electrode spacing: 2mm) of RA was screened for focal activation patterns (FAPs) potentially arising from SAN activation (SN-FAP). Conduction time, endo-epicardial delay and electrogram morphology (R/S-ratio) were described in the surrounding 1cm of SN-FAPs. Areas of conduction block (CB) were defined as conduction delays of ≥12ms and endo-epicardial asynchrony (EEA) as activation time differences of exact opposite electrodes of ≥15ms. Results In 20 patients (75% male, 66±7 years) a total of 28 SN-FAPs with unique activation patterns were observed only in superior RA (n=22) and mid RA (n=6). Conduction velocity of SN-FAPs was 1.3m/s [0.8–2.5] and percentage of EEA was 5% [2–18]. Conduction velocity was lower when lines of CB were present within 6mm of the SN-FAPs (p=0.040). Significantly larger R-peaks were observed in mid RA compared to superior RA. (mid R/S-ratio:0.15 [0.067–0.34] vs. superior R/S-ratio:0.045 [0.026–0.062], p=0.004). Conclusions Conduction velocities around the SAN area are comparable to the remainder of the atria. SN-FAPs occurred in both superior and mid RA, suggesting presence of multiple exit sites of the SAN. Furthermore, inter-individual differences in rS-morphology indicate that SAN activation may originate from multiple layers within the atrial wall. Funding Acknowledgement Type of funding source: None

ATAC-Seq Reveals an Isl1 Enhancer That Regulates Sinoatrial Node Development and Function.

Cardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have a distinct gene expression program that allows them to fire automatically and initiate the heartbeat. Although critical SAN transcription factors, including Isl1 (Islet-1), Tbx3 (T-box transcription factor 3), and Shox2 (short-stature homeobox protein 2), have been identified, the cis-regulatory architecture that governs PC-specific gene expression is not understood, and discrete enhancers required for gene regulation in the SAN have not been identified. To define the epigenetic profile of PCs using comparative ATAC-seq (assay for transposase-accessible chromatin with sequencing) and to identify novel enhancers involved in SAN gene regulation, development, and function. We used ATAC-seq on sorted neonatal mouse SAN to compare regions of accessible chromatin in PCs and right atrial cardiomyocytes. PC-enriched assay for transposase-accessible chromatin peaks, representing candidate SAN regulatory elements, were located near established SAN genes and were enriched for distinct sets of TF (transcription factor) binding sites. Among several novel SAN enhancers that were experimentally validated using transgenic mice, we identified a 2.9-kb regulatory element at the Isl1 locus that was active specifically in the cardiac inflow at embryonic day 8.5 and throughout later SAN development and maturation. Deletion of this enhancer from the genome of mice resulted in SAN hypoplasia and sinus arrhythmias. The mouse SAN enhancer also directed reporter activity to the inflow tract in developing zebrafish hearts, demonstrating deep conservation of its upstream regulatory network. Finally, single nucleotide polymorphisms in the human genome that occur near the region syntenic to the mouse enhancer exhibit significant associations with resting heart rate in human populations. (1) PCs have distinct regions of accessible chromatin that correlate with their gene expression profile and contain novel SAN enhancers, (2) cis-regulation of Isl1 specifically in the SAN depends upon a conserved SAN enhancer that regulates PC development and SAN function, and (3) a corresponding human ISL1 enhancer may regulate human SAN function.

Contribution of estrogen to the pregnancy-induced increase in cardiac automaticity

BackgroundThe heart rate progressively increases throughout pregnancy, reaching a maximum in the third trimester. This elevated heart rate is also present in pregnant mice and is associated with accelerated automaticity, higher density of the pacemaker current If and changes in Ca2+ homeostasis in sinoatrial node (SAN) cells. Strong evidence has also been provided showing that 17β-estradiol (E2) and estrogen receptor α (ERα) regulate heart rate. Accordingly, we sought to determine whether E2 levels found in late pregnancy cause the increased cardiac automaticity associated with pregnancy. Methods and resultsVoltage- and current-clamp experiments were carried out on SAN cells isolated from female mice lacking estrogen receptor alpha (ERKOα) or beta (ERKOβ) receiving chronic E2 treatment mimicking late pregnancy concentrations. E2 treatment significantly increased the action potential rate (284 ± 24 bpm, +E2 354 ± 23 bpm, p = 0.040) and the density of If (+52%) in SAN cells from ERKOβ mice. However, If density remains unchanged in SAN cells from E2-treated ERKOα mice. Additionally, E2 also increased If density (+67%) in nodal-like human-induced pluripotent stem cell-derived cardiomyocytes (N-hiPSC-CM), recapitulating in a human SAN cell model the effect produced in mice. However, the L-type calcium current (ICaL) and Ca2+ transients, examined using N-hiPSC-CM and SAN cells respectively, were not affected by E2, indicating that other mechanisms contribute to changes observed in these parameters during pregnancy. ConclusionThe accelerated SAN automaticity observed in E2-treated ERKOβ mice is explained by an increased If density mediated by ERα, demonstrating that E2 plays a major role in regulating SAN function during pregnancy.

Etrasimod, a Selective S1P1,4,5 Receptor Modulator, Exhibits Differential G Protein Signaling Compared to Other S1P Receptor Modulators

BackgroundEtrasimod (APD334) is an oral, selective sphingosine‐1‐phosphate (S1P) receptor modulator in development for atopic dermatitis and inflammatory bowel disease. In humans, S1P receptor modulators decrease heart rate at first‐dose due to initial on‐target S1P receptor 1 (S1P1) agonism in cardiac tissue. S1P1 agonism activates Gi and β‐arrestin signaling pathways: β‐arrestin activation leads to receptor internalization, the proposed mechanism by which S1P receptor modulators regulate lymphocyte trafficking; Gi‐coupled signaling activates G‐protein coupled inwardly rectifying potassium (GIRK) channels, that may contribute to the regulation of heart rate. Approved or investigational S1P receptor modulators have demonstrated different degrees of heart rate reduction and atrioventricular block in clinical studies. Such differences may be the result of differential signaling through the Gi coupled GIRK pathway.AimTo investigate S1P receptor expression in human sinoatrial (SA) node tissue, and the relative S1P1 mediated signaling events and GIRK channel activation of etrasimod and other S1P receptor modulators in the context of first dose heart rate effects.MethodsS1P receptor profiling was performed by qPCR in normal human primary SA node and right atrium samples. Potencies for multiple S1P receptor modulators to activate different S1P1 downstream signaling pathways (GTPγS, cAMP, β‐arrestin, S1P1 internalization) were measured in recombinant human‐S1P1 expressing cells. Gi mediated inhibition of cAMP was also measured in primary HUVEC. GIRK channel activation was measured in isolated adult human primary atrial myocytes.ResultsProfiling of S1P receptors in normal human atrial tissue and SA node demonstrated enriched expression of S1P1,2,3 with only minor expression of S1P4,5. Etrasimod was equipotent to other S1P receptor‐ modulators such as ozanimod, fingolimod, and siponimod in promoting β‐arrestin recruitment and S1P1 receptor internalization. Etrasimod was significantly less potent than ozanimod, fingolimod, and siponimod in assays measuring G‐protein activation (GTPγS binding and cAMP inhibition). In comparisons of GIRK activation, etrasimod was found to be a less potent activator of human atrial GIRK channels than ozanimod.ConclusionsWhen compared to other S1P1 modulators, the pharmacological profile of etrasimod at the S1P1 receptor suggests diminished capacity for signaling through Gi‐protein dependent pathways believed to be responsible for transient reductions in heart rate, while maintaining potent signaling in the β‐arrestin recruitment and receptor internalization related pathways underpinning the therapeutic efficacy of S1P1 modulators in immune‐mediated inflammatory disease settings.Support or Funding InformationThe study was sponsored by Arena Pharmaceuticals, Inc.

PHARMACOGENETICS AND DRUG DISCOVERY FOR ANTHRACYCLINE-INDUCED CARDIOTOXICITY ENABLED BY SINOATRIAL NODE-LIKE CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS

The sinoatrial node (SAN) is the primary pacemaker of the heart. The human SAN is poorly understood due to limited primary tissue access and lack of robust in vitro derivation methods. We developed an efficient strategy, using a dual SHOX2:GFP; MYH6:mCherry knock-in reporter line, to generate and

Impaired neuronal sodium channels cause intranodal conduction failure and reentrant arrhythmias in human sinoatrial node

Mechanisms for human sinoatrial node (SAN) dysfunction are poorly understood and whether human SAN excitability requires voltage-gated sodium channels (Nav) remains controversial. Here, we report that neuronal (n)Nav blockade and selective nNav1.6 blockade during high-resolution optical mapping in explanted human hearts depress intranodal SAN conduction, which worsens during autonomic stimulation and overdrive suppression to conduction failure. Partial cardiac (c)Nav blockade further impairs automaticity and intranodal conduction, leading to beat-to-beat variability and reentry. Multiple nNav transcripts are higher in SAN vs atria; heterogeneous alterations of several isoforms, specifically nNav1.6, are associated with heart failure and chronic alcohol consumption. In silico simulations of Nav distributions suggest that INa is essential for SAN conduction, especially in fibrotic failing hearts. Our results reveal that not only cNav but nNav are also integral for preventing disease-induced failure in human SAN intranodal conduction. Disease-impaired nNav may underlie patient-specific SAN dysfunctions and should be considered to treat arrhythmias.

In silico study of the effects of anti-arrhythmic drug treatment on sinoatrial node function for patients with atrial fibrillation

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 (If) 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 INa, IKur, ICaL, ICaT, If 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.