Sinoatrial Node Pacemaker Research Articles

Caveolar Compartmentalization of Pacemaker Signaling is Required for Stable Rhythmicity of Sinus Nodal Cells and is Disrupted in Heart Failure.

Heart rhythm relies on complex interactions between electrogenic membrane proteins and intracellular Ca2+ signaling in sinoatrial node (SAN) myocytes; however, mechanisms underlying the functional organization of proteins involved in SAN pacemaking and its structural foundation remain elusive. Caveolae are nanoscale, plasma membrane pits that compartmentalize various ion channels and transporters, including those involved in SAN pacemaking, via binding with the caveolin-3 scaffolding protein, but the precise role of caveolae in cardiac pacemaker function is unknown. Our objective was to determine the role of caveolae in SAN pacemaking and dysfunction (SND). Biochemical co-purification, in vivo electrocardiogram monitoring, ex vivo optical mapping, in vitro confocal Ca2+ imaging, and immunofluorescent and electron microscopy analyses were performed in wild type, cardiac-specific caveolin-3 knockout, and 8-weeks post-myocardial infarction heart failure (HF) mice. SAN tissue samples from donor human hearts were used for biochemical studies. We utilized a novel 3-dimensional single SAN cell mathematical model to determine the functional outcomes of protein nanodomain-specific localization and redistribution in SAN pacemaking. In both mouse and human SANs, caveolae compartmentalized HCN4, Cav1.2, Cav1.3, Cav3.1 and NCX1 proteins within discrete pacemaker signalosomes via direct association with caveolin-3. This compartmentalization positioned electrogenic sarcolemmal proteins near the subsarcolemmal sarcoplasmic reticulum (SR) membrane and ensured fast and robust activation of NCX1 by subsarcolemmal local SR Ca2+ release events (LCRs), which diffuse across ~15-nm subsarcolemmal cleft. Disruption of caveolae led to the development of SND via suppression of pacemaker automaticity through a 50% decrease of the L-type Ca2+ current, a negative shift of the HCN current (I f) activation curve, and a 40% reduction of Na+/Ca2+-exchanger function, along with ~2.3-times widening of the sarcolemma-SR distance. These changes significantly decreased the SAN depolarizing force, both during diastolic depolarization and upstroke phase, leading to bradycardia, sinus pauses, recurrent development of SAN quiescence, and significant increase in heart rate lability. Computational modeling, supported by biochemical studies, identified NCX1 redistribution to extra-caveolar membrane as the primary mechanism of SAN pauses and quiescence due to the impaired ability of NCX1 to be effectively activated by LCRs and trigger action potentials. HF remodeling mirrored caveolae disruption leading to NCX1-LCR uncoupling and SND. SAN pacemaking is driven by complex protein interactions within a nanoscale caveolar pacemaker signalosome. Disruption of caveolae leads to SND, potentially demonstrating a new dimension of SAN remodeling and providing a newly recognized target for therapy.

Differentiation of Sinoatrial-like Cardiomyocytes as a Biological Pacemaker Model.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are widely used for disease modeling and pharmacological screening. However, their application has mainly focused on inherited cardiopathies affecting ventricular cardiomyocytes, leading to extensive knowledge on generating ventricular-like hiPSC-CMs. Electronic pacemakers, despite their utility, have significant disadvantages, including lack of hormonal responsiveness, infection risk, limited battery life, and inability to adapt to changes in heart size. Therefore, developing an in vitro multiscale model of the human sinoatrial node (SAN) pacemaker using hiPSC-CM and SAN-like cardiomyocyte differentiation protocols is essential. This would enhance the understanding of SAN-related pathologies and support targeted therapies. Generating SAN-like cardiomyocytes offers the potential for biological pacemakers and specialized conduction tissues, promising significant benefits for patients with conduction system defects. This review focuses on arrythmias related to pacemaker dysfunction, examining protocols' advantages and drawbacks for generating SAN-like cardiomyocytes from hESCs/hiPSCs, and discussing therapeutic approaches involving their engraftment in animal models.

Abstract We045: Development of SA Node Organoids: A Multicellular Approach to Biological Pacemaking

The rising prevalence of sinoatrial (SA) node dysfunction, attributed to the aging population, is emerging as a critical health issue. The SA node, a natural organoid within the heart, comprises the pacemaker cells embedded within a connective tissue matrix, insulated electrically from the surrounding atrial muscle cells. This isolated architecture, along with a heterogeneous cellular composition, is crucial for the SA node's robust pacemaking and electrical conduction. Developing methods to reconstruct this crucial heterogeneous structure could offer a long-term solution for individuals with SA node dysfunction. However, the use of current biological pacemakers, which typically involve a single cell type, has often led to unstable heart rhythms during one-month in vivo integration, challenging their long-term clinical application as biological pacemakers. To address existing SA node model limitations, we developed a SA node organoid that reproduces the human SA node's multicellular tissue structure. Initially, we developed a protocol for generating cardiac SA node organoids by employing a lentivirus to deliver dox-inducible genes for key pacemaker transcription factors, specifically TBX-18 or Shox-2. We mixed wild-type human-induced pluripotent stem cells (hiPSCs) with those modified to express TBX-18 or Shox-2. These cell mixtures were aggregated, embedded in a diluted Matrigel, and then exposed to a cardiac differentiation protocol, activating TBX-18 or Shox-2 at specific times (D1 or D7) via Dox administration. By D9, these organoids began to exhibit contraction, which lasted beyond 60 days, with notable upregulation of two transcription factors and pacemaker-specific genes following Dox treatment. Subsequently, we integrated the SA node organoid into 2D hiPSC-derived atrial muscle tissue constructs, maintaining geometric isolation within the cardiac tissue to enhance source-sink mismatch. This isolation allowed the SA node organoid to successfully trigger activity in adjacent dormant cardiomyocytes, preserving the rhythm for over 60 days. These findings could redefine the role of tissue architecture and cell diversity in the SA node's pacemaking function, potentially leading to a high-fidelity, tissue-level biological pacemaker as a new treatment strategy for SA node dysfunctions.

α1-Adrenergic Receptors Control the Activity of Sinoatrial Node by Modulating Transmembrane Transport of Chloride Anions

Norepinephrine (NE), which is released by sympathetic nerve endings, causes an increase in the frequency of spontaneous action potentials in the pacemaker cardiomyocytes of the sinoatrial node (SAN), also known as the “pacemaker” of the heart. This results in an increase in heart rate (HR). It is known that two types of postsynaptic adrenoreceptors (ARs), α1-AR and β-AR, can mediate the effects of NE. The role of α1-AR in the sympathetic control of heart rate and SAN automaticity, as well as the membrane mechanisms mediation the effects of α1-AR on the pacemaker, have not yet been elucidated. In this study, we utilized immunofluorescence confocal microscopy to examine the distribution of α1A-AR in the SAN of rats. Additionally, we assessed the expression of α1A-AR mRNA in the SAN tissue using RT-PCR. Furthermore, we investigated the impact of α1-AR stimulation on key functional parameters of the pacemaker, including the corrected sinus node recovery time (SNRT/cSNRT) and the SAN accommodation, using the Langendorff perfused heart technique. We also used optical mapping of the electrical activity of perfused, isolated tissue preparations to study the effect of α1-AR stimulation on the spatiotemporal characteristics of SAN excitation. We tested the effects of chloride transmembrane conductance blockade on alteration of functional parameters and pattern of SAN excitation caused by α1-AR. Fluorescent signals corresponding to α1A-AR have been identified in SAN cardiomyocytes, indicating the presence of α1A-AR at protein level. The expression of α1A-AR in SAN has been also confirmed at the mRNA level. The stimulation of α1-AR affects SAN functioning Phenylephrine (PHE) utilized as α1A-AR agonist causes a decrease in SNRT/cSNRT, as well as an acceleration of SAN accommodation. These effects were rate dependent and were observed at a high frequency of pacemaker tissue stimulation. PHE induces changes in the excitation pattern of the SAN. The effects of PHE on functional parameters and SAN excitation pattern are attenuated by Ca2+-dependent chloride channel blocker NPPB but remains unaffected by the protein kinase C inhibitor BIM. Our results suggest that cardiac α1-ARs are important for maintaining function of SAN pacemaker at high heart rates and that α1-AR signalling cascades in the SAN target Ca2+-dependent chloride channels are involved in the α1-adrenergic modulation of the electrophysiological properties of the heart pacemaker.

Cholecystokinin 1 Receptor Is Activated Permanently in Photodynamic Action

In contrast to agonist activation that is reversed after washout of agonist, we have found that cholecystokinin 1 receptor (CCK1R) is activated permanently by singlet oxygen in type II photodynamic action, the G Protein Coupled Receptor (GPCR) Activated By Singlet Oxygen is named GPCR-ABSO. CCK1R ectopically expressed in cell lines is activated in photodynamic action with photosensitizer sulphonated aluminum phthalocyanine (SALPC) or with genetically encoded protein photosensitizers (such as miniSOG, KillerRed) to trigger persistent calcium oscillations which are not washed out. Such permanent photodynamic CCK1R activation is seen also in freshly isolated pancreatic acini, which is inhibited by CCK1R antagonist. Photodynamic CCK1R activation is physiological in intensity, triggering calcium oscillations similar in frequencies to those induced by physiological CCK concentrations (10 - 100 pM). The molecular mechanisms for such permanent photodynamic activation of CCK1R at the physiological level are three-fold. When membrane proteins prepared from isolated rat pancreatic acini are subject to Western blot, CCK1R is found to be present as both dimers and monomers. After photodynamic action with SALPC at an intensity that would trigger persistent calcium oscillations in live cells, the resting receptor dimer is converted quantitatively to monomer, similar to agonist stimulated CCK1R monomerization revealed by bioluminescence resonance energy transfer (BRET) method in rat CCK1R-expressing COS cells, suggesting that photodynamic CCK1R activation we have demonstrated is likely quite physiological. Whilst CCK1R is activated by singlet oxygen in photodynamic action, the M3 acetylcholine receptor is not affected. However, when TM3 of CCK1R is transplanted to M3R to replace TM3 of M3R, the chimeric receptor M3R-TM3CCK1R becomes activatable in photodynamic action, after tagging with miniSOG. The calculated extent of miniSOG photodynamic activation of M3R, chimeric M3R-TM3CCK1R, and CCK1R are 0%, 65%, 100% respectively. Comparative studies with pancreatic acini isolated from the rat, mouse and Peking duck found that sensitivity for photodynamic CCK1R activation is, in descending order: mouse > rat > Peking duck, the same order of the size of structure-free region of intracellular loop 3 (ICL3). Other than CCK1R, CCK2R is photodynamically activated also, after tagging with protein photosensitizer miniSOG. CCK1R and CCK2R are known to be involved in vital physiological functions: pancreatic secretion, satiety sensation, sinoatrial node pace-making, memory formation and storage, and others. Our work implies that all these and other physiological functions might be modulated in vivo by photodynamic CCK1R activation. The fact that such property of permanent photodynamic activation could be transplanted from CCK1R to other GPCR such as M3R suggests that photodynamic activation of GPCR could be used to tease apart the roles and functions of vast numbers of GPCR. Other functional proteins such as NAD(P)H oxidase 2, BK/Slo channel, CaMKII, riboflavin transporter (SLC52A1/RFVT1), might also be photodynamically activated permanently. This work was supported by The Natural Science Foundation of China (NSFC, Grant Nos. 31971170 and 32271278). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ivabradine in Septic Shock: A Narrative Review.

In patients with septic shock, compensatory tachycardia initially serves to maintain adequate cardiac output and tissue oxygenation but may persist despite appropriate fluid and vasopressor resuscitation. This sustained elevation in heart rate and altered heart rate variability, indicative of autonomic dysfunction, is a well-established independent predictor of adverse outcomes in critical illness. Elevated heart rate exacerbates myocardial oxygen demand, reduces ventricular filling time, compromises coronary perfusion during diastole, and impairs the isovolumetric relaxation phase of the cardiac cycle, contributing to ventricular-arterial decoupling. This also leads to increased ventricular and atrial filling pressures, with a heightened risk of arrhythmias. Ivabradine, a highly selective inhibitor of the sinoatrial node's pacemaker current (If or "funny" current), mitigates heart rate by modulating diastolic depolarization slope without affecting contractility. By exerting a selective chronotropic effect devoid of negative inotropic properties, ivabradine shows potential for improving hemodynamics in septic shock patients with cardiac dysfunction. This review evaluates the plausible mechanisms and existing evidence regarding the utility of ivabradine in managing patients with septic shock.

Pathogenicity of de novo CACNA1D Ca2+ channel variants predicted from sequence co-variation

Voltage-gated L-type Cav1.3 Ca2+ channels support numerous physiological functions including neuronal excitability, sinoatrial node pacemaking, hearing, and hormone secretion. De novo missense mutations in the gene of their pore-forming α1-subunit (CACNA1D) induce severe gating defects which lead to autism spectrum disorder and a more severe neurological disorder with and without endocrine symptoms. The number of CACNA1D variants reported is constantly rising, but their pathogenic potential often remains unclear, which complicates clinical decision-making. Since functional tests are time-consuming and not always available, bioinformatic tools further improving pathogenicity potential prediction of novel variants are needed. Here we employed evolutionary analysis considering sequences of the Cav1.3 α1-subunit throughout the animal kingdom to predict the pathogenicity of human disease-associated CACNA1D missense variants. Co-variation analyses of evolutionary information revealed residue–residue couplings and allowed to generate a score, which correctly predicted previously identified pathogenic variants, supported pathogenicity in variants previously classified as likely pathogenic and even led to the re-classification or re-examination of 18 out of 80 variants previously assessed with clinical and electrophysiological data. Based on the prediction score, we electrophysiologically tested one variant (V584I) and found significant gating changes associated with pathogenic risks. Thus, our co-variation model represents a valuable addition to complement the assessment of the pathogenicity of CACNA1D variants completely independent of clinical diagnoses, electrophysiology, structural or biophysical considerations, and solely based on evolutionary analyses.

Mechanistic insight into the functional role of human sinoatrial node conduction pathways and pacemaker compartments heterogeneity: A computer model analysis.

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.

Age-related structural and functional changes of the intracardiac nervous system

BackgroundAlthough aging is known to be associated with an increased incidence of both atrial and ventricular arrhythmias, there is limited knowledge about how Schwann cells (SC) and the intracardiac nervous system (iCNS) remodel with age. Here we investigate the differences in cardiac SC, parasympathetic nerve fibers, and muscarinic acetylcholine receptor M2 (M2R) expression in young and old mice. Additionally, we examine age-related changes in cardiac responses to sympathomimetic and parasympathomimetic drugs. Methods and resultsLower SC density, lower SC proliferation and fewer parasympathetic nerve fibers were observed in cardiac and, as a control sciatic nerves from old (20–24 months) compared to young mice (2–3 months). In old mice, chondroitin sulfate proteoglycan 4 (CSPG4) was increased in sciatic but not cardiac nerves. Expression of M2R was lower in ventricular myocardium and ventricular conduction system from old mice compared to young mice, while no significant difference was seen in M2R expression in sino-atrial or atrio-ventricular node pacemaker tissue. Heart rate was slower and PQ intervals were longer in Langendorff-perfused hearts from old mice. Ventricular tachycardia and fibrillation were more frequently observed in response to carbachol administration in hearts from old mice versus those from young mice. ConclusionsOn the background of reduced presence of SC and parasympathetic nerve fibers, and of lower M2R expression in ventricular cardiomyocytes and conduction system of aged hearts, the propensity of ventricular arrhythmogenesis upon parasympathomimetic drug application is increased. Whether this is caused by an increase in heterogeneity of iCNS structure and function remains to be elucidated.

Single-Cell Atlas of Neonatal Mouse Hearts Reveals an Unexpected Cardiomyocyte.

Single-cell RNA sequencing is widely used in cancer research and organ development because of its powerful ability to analyze cellular heterogeneity. However, its application in cardiomyocytes is dissatisfactory mainly because the cardiomyocytes are too large and fragile to withstand traditional single-cell approaches. Through designing the isolation procedure of neonatal mouse cardiac cells, we provide detailed cellular atlases of the heart at single-cell resolution across 4 different stages after birth. We have obtained 10 000 cardiomyocytes; to our knowledge, this is the most extensive reference framework to date. Moreover, we have discovered unexpected erythrocyte-like cardiomyocyte-terminal cardiomyocytes, comprising more than a third of all cardiomyocytes. Only a few genes are highly expressed in these cardiomyocytes. They are highly differentiated cardiomyocytes that function as contraction pumps. In addition, we have identified 2 cardiomyocyte-like conducting cells, lending support to the theory that the sinoatrial node pacemaker cells are specialized cardiomyocytes. Notably, we provide an initial blueprint for comprehensive interactions between cardiomyocytes and other cardiac cells. This mouse cardiac cell atlas improves our understanding of cardiomyocyte heterogeneity and provides a valuable reference in response to varying physiological conditions and diseases.

A spatially-resolved multiomic cell atlas reveals gene regulatory networks underlying cell specification in the developing human heart

Abstract Introduction Cardiogenesis requires the differentiation and coordinated function of a large variety of cell types. Congenital heart disease (CHD), affecting 1% of live births[1], and several adult-onset heart diseases result when these processes go awry[2–4]. To understand the cellular and molecular mechanisms at play we must define the repertoire of cell types in the developing heart as well as the cellular niches within which they interact. Single-cell RNA sequencing is a powerful means of defining cell populations, however existing studies of the human foetal heart[5,6] have been hampered by low cell numbers which limits detection of rare but important cell types. To create an improved atlas we increased the number of cells, increased the age range of foetal hearts sampled, and generated spatial transcriptomic data to map the defined cells to multicellular niches of co-located and interacting cells. To better infer the transcription factors (TFs) governing cell identity using gene regulatory network (GRN) inference we also generated paired single-cell resolution RNA and chromatin accessibility profiles using Assay for Transposase-Accessible Chromatin (ATAC) sequencing. Methods We performed paired single-nuclei RNA and ATAC sequencing (10X Multiome) and single-cell RNAseq (10X 3’ RNAseq) on 23 human foetal hearts ranging from 4 to 20 post-conception weeks. In addition we generated spatial transcriptomic data (10X Visium) from these hearts. We validated cell types defined in single-cell data using protein-level immunofluorescence imaging. Results Our healthy reference atlas includes 300k cells and nuclei. Using their gene expression profile we resolved 46 fine-grained cell types (FIGURE 1). We used cell2location[7], a machine learning classifier trained on the atlas to map the defined cell types to the spatial data (FIGURE 2) and detected niches of co-locating cells, such as a macrophage population in the walls of the great vessels predicted to interact with mural cells. We were able to define location-specific transcriptional signatures such as cardiomyocytes of each chamber (as well as the compact and trabeculated myocardium) and smooth muscle cells specific to great vessels vs coronary arteries. Using a combination of RNA and ATAC data we inferred cell-specific GRNs, which implicated novel TFs such as MSX2 in sinoatrial node pacemaker cells and the enrichment of CHD genes amongst cardiomyocyte-specific GRNs. In the spatial data we showed that gene modules associated with different forms of CHD enrich in specific anatomical regions, such as the outflow tract. Discussion: Together these data constitute the most comprehensive cardiac developmental cell atlas to date. It sheds new light on the diversity of cell states, their niches, and their vulnerability to the genetic causes of CHD.Figure 1:UMAP showing atlasFigure 2:Spatial transcriptomic mapping

Abstract 19064: Sex Specific Gene Profile in Canine Sinoatrial Node

Introduction: Biological sex plays an important role in cardiac function and occurrence of cardiac arrhythmias. Clinical reports show that adult females have faster resting and intrinsic heart rate (HR) while males have faster HR peak and larger HR reserve. Furthermore, inappropriate sinus tachycardia primarily occurs in females, suggesting sex-specific function and/or structure of the sinoatrial node (SAN). The aim of the study is to define sex-specific molecular features of the SAN complex. Methods: mRNA was isolated from SAN pacemaker tissue of healthy adult canines (male=4, female=4). Differential RNA expression patterns of >400 genes, including fibrosis, inflammation, and metabolism markers were quantified with nCounter Nanostring fibrosis panel. Quantitative PCR was used to compare mRNAs of key ion channels related to SAN pacemaker function. Results: Nanostring panel revealed sex-specific and SAN-specific gene profiles of inflammation and fibrotic markers with multiple mRNAs higher in male (e.g. NLRP3, CD86, COL3A1, TGFB1) and several higher in females (e.g. MARCOL, IL20RB). mRNAs responsible for SAN pacemaking e.g. HCN1, HCN4, Cav1.3, and GIRK4 are higher expressed in female SAN. Sex-specific retrospective analysis of published gene profiles from non-failing human hearts showed a similar pattern to canine with HCN1, HCN4, Cav1.3 expression higher in female SAN. Conclusions: The study revealed significantly different gene expressions of fibrosis, inflammation, and pacemaking in male vs female SAN. The higher expression of HCN1/4 and Cav1.3 may explain the faster intrinsic sinus rhythm and susceptibility to SAN arrhythmias in females. The elevated fibrosis and inflammation genes in male SAN may suggest higher vulnerability to fibrotic remodeling during cardiac disease.

Abstract 13327: Mechanistic Basis of Sinoatrial Node Pacemaker Failure in Atrial Cardiomyopathy

Introduction: Sinoatrial node (SAN) dysfunction commonly occurs in atrial cardiomyopathy (ACM: “tachy-brady syndrome”), often requiring pacemaker implantation, but mechanisms are unknown. Atrial-selective myosin light chain-4 (MYL-4) mutations cause familial ACM with a high rate of early SAN dysfunction and pacemaker requirement. We studied rats engineered with a knock-in MYL-4 clinical E11K mutation and addressed mechanisms of SAN dysfunction. Methods: Atrial electrical properties were assessed with in vivo electrophysiology (EPS), ex vivo optical mapping, SAN single-cell voltage and current clamp, Ca 2+ -handling with Ca 2+ -sensitive dye microscopy. Gene expression was assessed by qPCR and fibrosis with Masson-Trichrome stain. Results: E11K rats showed progressive SAN recovery-time increases (Fig. A) and conduction-slowing (Fig. B). SAN cells showed slowed phase-4 depolarization (Fig. C) and reduced pacemaker “funny” current (I f ) (Fig. D). Ca 2+ homeostasis was also affected, showing decreased Ca 2+ -transients (Fig. E). SAN also displayed remodeling with increased collagen deposition (Fig. F). Conclusions: ACM resulting from E11K mutation induces multimodal electrical and structural remodeling of the SAN region, worsening with age. These observations provide new insights into mechanisms of the clinically important tachy-brady syndrome associated with ACM.

Lipopolysaccharide-induced sepsis impairs M2R-GIRK signaling in the mouse sinoatrial node

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 Ca2+ 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 IKACh activation in SAN cells, reduction in Ca2+ 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.

BS-452759-2 HIGH-RESOLUTION 3D MULTIMODALITY IMAGING TO DISTINGUISH INTRAMURAL STRUCTURE OF HUMAN SINOATRIAL NODE VS EPI AND ENDO ATRIAL EXITS

Defining and targeting the human sinoatrial node (SAN) is challenging since clinical electrode mapping can visualize only surface epi/endocardial (Epi/Endo) exits of SAN activation through conduction pathways and not the leading pacemaker within the 3D intramural structure. To distinguish the functional-structural features of the human SAN pacemaker vs Epi-Endo exits by 3D multimodality imaging. Simultaneous EpiEndo dual-sided near infrared transmural optical mapping (NIOM, 300-500μm2 resolution) was conducted on coronary perfused explanted human atria (n=5, 45±20 y.o.). SAN pacemaker and exit sites were defined by NIOM activation maps. Histology-validated contrast-enhanced MRI (CE-MRI, ∼100μm3 resolution) was used to quantitatively analyze the 3D atrial structure of the SAN pacemaker vs exit sites, including wall thickness, transmural fiber orientation and fibrosis content in the sub-Epi, intramural, and sub-Endo layers. All ex-vivo preparations had stable sinus rhythm (SR, 77±14 bpm) at baseline with leading pacemakers in the intramural SANpacemaker compartments. In total, 24 SR recordings at baseline and autonomic stimulations) were analyzed. NIOM detected 29 exits on Epi and 32 exits on Endo. The majority of Epi or Endo earliest exits (88%) were distributed on the CT while secondary exits were located along the CT border. The most distant superior, middle, and inferior exits were 14.1±2.4mm, 8.8±6.3mm, and 12.3±3.1mm away from the SAN pacemakers respectively. 3D quantitative structural analysis revealed that transmural wall thickness is significantly less across the SAN pacemaker than atrial exit sites, but the fibrosis content of intramural and sub-Endo layers is significantly higher in the SAN than in exit sites. There are transmural myofibers that were defined primarily at the exit sites along CT but not within SAN. This study provides a quantitative atlas of the 3D architecture of the human SAN, and reveals that SAN pacemakers can be distinguished from both Epi/Endo exits by wall thickness, intramural/sub-Endo fibrosis content, and transmural fiber orientations. The new 3D transmural imaging analysis approach can be integrated with clinical electrode mapping, which may help to define SAN pacemaker and guide patient-specific targeted ablation of atrial SAN and CT arrhythmias.

Control of the fight-or-flight response of heart rate by L-type Cav1.3 (α1D) calcium channels

The mechanisms underlying the increase in the frequency of heartbeat induced by catecholamines, so called fight-or-flight response (FFR) of heart rate (HR), are incompletely understood. cAMP-dependent regulation of f-(HCN) channels, underlying the “funny” current (If), and ryanodine receptor 2 (RyR2)-mediated Ca2+ release are considered as the predominant mechanisms of HR FFR. However, it is showed that If contributes to setting the basal HR, but is not required for the sympathetic regulation of pacemaking. In addition, impairment of RyR2-mediated [Ca2+]i release by ryanodine only reduces β-adrenergic regulation of pacemaker activity, suggesting that other mechanism(s) are involved in the generation of HR FFR. We have shown that L-type Cav1.3 calcium channels are required for β-adrenergic mediated initiation of firing in dormant sino-atrial node (SAN) pacemaker cells. To define the role of Cav1.3 channels in the FFR of HR and SAN automaticity. We crossed mice deficient in Cav1.3 channels (Cav1.3-/-) with mice (CNBD) lacking cAMP sensitivity of f-channels. In vivo and in isolated hearts ECG recordings were performed. Automaticity and diastolic current of SAN cells were evaluated using patch-clamp technique. Cav1.3-/- and CNBD mice displayed bradycardia at rest, but similar degree of increase in HR after β-adrenergic stimulation by injection of isoproterenol (ISO) in comparison to WT mice. However, Cav1.3-/-/CNBD mice did not show significant HR increase in vivo after injection of ISO or during physical exercise. ISO failed to increase the rate in isolated herats and in the SAN cells from Cav1.3-/-/CNBD mice. Consistent with these data, mutant mice in which L-type Cav1.2 channels have been rendered insensitive to dihydropyridines (Cav1.2DHP-/-) showed that concomitant selective pharmacologic inhibition of Cav1.3 and cAMP-dependent dependent regulation of f-HCN channels by the di-cyclic nucleotide di-cGMP prevented the positive chronotropic response to ISO of SAN cells automaticity. Under the same conditions, ISO did not significantly increase diastolic current, explaining loss of SAN FFR. Our study identifies L-type Cav1.3 channels as key effectors of β-adrenergic regulation of SAN automaticity with If.

What makes the sinoatrial node tick? A question not for the faint of heart.

Even before the sinoatrial node (SAN) was discovered, cardiovascular science was engaged in an active investigation of when and why the heart would beat. After the electrochemical theory of bioelectric membrane potentials was formulated and the first action potentials were measured in contracting muscle cells, the field became divided: some investigators studied electrophysiology and ion channels, others studied muscle contraction. It later became known that changes in intracellular Ca2+ cause contraction. The pacemaking field was reunited by the coupled-clock theory of pacemaker cell function, which integrated intracellular Ca2+ cycling and transmembrane voltage into one rhythmogenic system. In this review, we will discuss recent discoveries that contextualize the coupled-clock system, first described in isolated SAN cells, into the complex world of SAN tissue: heterogeneous local Ca2+ releases, generated within SAN pacemaker cells and regulated by the other cell types within the SAN cytoarchitecture, variably co-localize and synchronize to give rise to relatively rhythmic impulses that emanate from the SAN to excite the heart. We will ultimately conceptualize the SAN as a brain-like structure, composed of intercommunicating meshworks of multiple types of pacemaker cells and interstitial cells, intertwined networks of nerves and glial cells and more. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Human Sinoatrial Node Pacemaker Activity: Role of the Slow Component of the Delayed Rectifier K+ Current, IKs.

The pacemaker activity of the sinoatrial node (SAN) has been studied extensively in animal species but is virtually unexplored in humans. Here we assess the role of the slowly activating component of the delayed rectifier K+ current (IKs) in human SAN pacemaker activity and its dependence on heart rate and β-adrenergic stimulation. HEK-293 cells were transiently transfected with wild-type KCNQ1 and KCNE1 cDNA, encoding the α- and β-subunits of the IKs channel, respectively. KCNQ1/KCNE1 currents were recorded both during a traditional voltage clamp and during an action potential (AP) clamp with human SAN-like APs. Forskolin (10 µmol/L) was used to increase the intracellular cAMP level, thus mimicking β-adrenergic stimulation. The experimentally observed effects were evaluated in the Fabbri-Severi computer model of an isolated human SAN cell. Transfected HEK-293 cells displayed large IKs-like outward currents in response to depolarizing voltage clamp steps. Forskolin significantly increased the current density and significantly shifted the half-maximal activation voltage towards more negative potentials. Furthermore, forskolin significantly accelerated activation without affecting the rate of deactivation. During an AP clamp, the KCNQ1/KCNE1 current was substantial during the AP phase, but relatively small during diastolic depolarization. In the presence of forskolin, the KCNQ1/KCNE1 current during both the AP phase and diastolic depolarization increased, resulting in a clearly active KCNQ1/KCNE1 current during diastolic depolarization, particularly at shorter cycle lengths. Computer simulations demonstrated that IKs reduces the intrinsic beating rate through its slowing effect on diastolic depolarization at all levels of autonomic tone and that gain-of-function mutations in KCNQ1 may exert a marked bradycardic effect during vagal tone. In conclusion, IKs is active during human SAN pacemaker activity and has a strong dependence on heart rate and cAMP level, with a prominent role at all levels of autonomic tone.

Disrupting methyl-CpG-binding protein 2 expression induces the transformation of induced pluripotent stem cell cardiomyocytes into pacemaker-like cells by insulin gene enhancer binding protein 1.

The experiment will explore whether interfering with the expression of methyl-CpG-binding protein 2 (MecP2) can enhance the ability of insulin gene enhancer binding protein 1 (ISL1) to induce iPSC-CMs to differentiate into pacemaker-like cells. Differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes (CMs) can be induced via the regulation of the Wnt signaling pathway. Real-time quantitative PCR (qPCR), western blotting, immunofluorescence staining, and patch-clamp technique were used to analyze the ability of ISL1 to induce the transformation of iPSC-CMs into pacemaker-like cells. Calcium spark, patch-clamp technique, and real-time qPCR were used to verify whether disrupting the expression of MeCP2 enhanced this ability of ISL1 to induce the differentiation of iPSC-CMs into pacemaker cells. Transplant pacemaker-like cardiomyocytes into the myocardium of mice to observe whether the pacemaker cells can survive in the tissue for a long time. RT-qPCR and patch-clamp analyses showed that overexpression of ISL1 induced the successful differentiation of iPSC-CMs into pacemaker cells. ISL1-overexpressing pacemaker-like cells possessed typical characteristics of pacemaker morphology, including action potential and If inward current. Chromatin immunoprecipitation results showed that MeCP2 bound to the promoter region of HCN4. Following disruption of MeCP2 expression, the gene expression of sinoatrial node-specific transcription factors, If inward current, and cardiac rhythm changes in iPSC-CMs resembled those of sinoatrial node pacemaker cells. Therefore, ISL1 induced the differentiation of iPSC-CMs into pacemaker-like cells, and knockdown of MeCP2 increased this effect. Frozen section results showed that surviving pacemaker-like cells could still be observed in myocardial tissue after 45 days. Experiments have found that interfering with the expression of MeCP2 can increase the ability of ISL1 to induce iPSC-CM cells to differentiate into pacemaker-like cells. And the pacemaker-like cells obtained in this experiment can survive in myocardial tissue for a long time.

Synergy between Membrane Currents Prevents Severe Bradycardia in Mouse Sinoatrial Node Tissue

Bradycardia is initiated by the sinoatrial node (SAN), which is regulated by a coupled-clock system. Due to the clock coupling, reduction in the 'funny' current (If), which affects SAN automaticity, can be compensated, thus preventing severe bradycardia. We hypothesize that this fail-safe system is an inherent feature of SAN pacemaker cells and is driven by synergy between If and other ion channels. This work aimed to characterize the connection between membrane currents and their underlying mechanisms in SAN cells. SAN tissues were isolated from C57BL mice and Ca2+ signaling was measured in pacemaker cells within them. A computational model of SAN cells was used to understand the interactions between cell components. Beat interval (BI) was prolonged by 54 ± 18% (N = 16) and 30 ± 9% (N = 21) in response to If blockade, by ivabradine, or sodium current (INa) blockade, by tetrodotoxin, respectively. Combined drug application had a synergistic effect, manifested by a BI prolonged by 143 ± 25% (N = 18). A prolongation in the local Ca2+ release period, which reports on the level of crosstalk within the coupled-clock system, was measured and correlated with the prolongation in BI. The computational model predicted that INa increases in response to If blockade and that this connection is mediated by changes in T and L-type Ca2+ channels.