Sinus Node Research Articles

Beta-Blocker in Heart Rate Control and Cardio Protection: The Role of ADRB1 Variants and HCN4 Regulation – A Systematic Review

Elevated heart rate is linked to adverse cardiovascular outcomes. Sinoatrial (SA) nodes, hyperpolarization-activated cyclic nucleotide-gated-4 (HCN4) channels, and beta1-adrenergic receptor (ADRB1) are responsible for generating the heart rate. Beta-blockers have a cardioprotective effect on heart failure, including controlling heart rate. However, the responses to beta-blockers can vary among individuals. ADRB1 genetic variability may be contributed to the differential beta-blocker effect in heart failure. HCN4 also performs a crucial function in the pacemaker cells of the heart. Exploring the effect of beta-blockers in pacemaker cells is expanding the view of their role and their therapeutic response in heart failure. The objectives of this study were to identify ADRB1 genetic variants affecting heart rate response in heart failure subjects with beta-blocker treatment and to explore the effect of beta-blockers on HCN4 channels and SA nodes. A systematic review was performed using three databases. Eight of 668 manuscripts were selected. The systematic review found that ADRB1 genetic variants (A145G (Ser49Gly) and C1165G (Arg389Gly)) can affect heart rate response in beta-blocker-treated heart failure. The study also found that the percentage of patients with the Ser49Ser-Gly389X haplotype achieved a heart rate target was higher than other haplotypes. Individuals with the Arg389Arg genotype necessitated a markedly increased amount of beta-blocker dose to reach the identical heart rate target compared to those with the Gly389X gene variation. In addition, the review found that carvedilol, a beta-blocker derivative, demonstrated beneficial effects in inhibiting HCN-gated channels. Bisoprolol and carvedilol improved channel regulation in the SA Node by reversing the downregulation of HCN4 and sodium channels. In general, this systematic review provides important insights into beta-blockers in treating heart failure, specifically concerning the genetic variability of ADRB1 and the beta-blockers effect on the SA node and HCN4 channels.

Feed Forward Modeling: an efficient approach for mathematical modeling of the force frequency relationship in the rabbit isolated ventricular myocyte

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 Ca2+ 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 (t50) 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.

Cardiac automaticity is modulated by IKACh in sinoatrial node during pregnancy.

Pregnant women have a significantly elevated resting heart rate (HR), which makes cardiac arrhythmias more likely to occur. Although electrical remodeling of the sinoatrial node (SAN) has been documented, the underlying mechanism is not fully understood. The acetylcholine-activated potassium current (IKACh), one of the major repolarizing currents in the SAN, plays a critical role in HR control by hyperpolarizing the maximal diastolic potential (MDP) of the SAN action potential (AP), thereby reducing SAN automaticity and HR. Thus, considering its essential role in cardiac automaticity, this study aims to determine whether changes in IKACh are potentially involved in the increased HR associated with pregnancy. Experiments were conducted on non-pregnant (NP, 2-3 months old) and pregnant (P, 17-18 gestation days) female CD-1 mice. IKACh was recorded on spontaneously beating SAN cells using the muscarinic agonist carbachol (CCh). Voltage-clamp data showed a reduction in IKACh density during pregnancy, which returned to control values shortly after delivery. The reduction in IKACh was explained by a decrease in protein expression of Kir3.1 channel subunit and the muscarinic type 2 receptor. In agreement with these findings, current-clamp data shows that the MDP of SAN cells from P mice were less hyperpolarized following CCh administration. Surface electrocardiograms (ECGs) recorded on anesthetized mice revealed that the cholinergic antagonist atropine and the selective KACh channel blocker tertiapin-Q increased HR in NP mice and had only a minimal effect on P mice. AP and ECG data also showed that pregnancy is associated with a decrease in beating and heart rate variability, respectively. IKACh function and expression are decreased in the mouse SAN during pregnancy, strongly suggesting that, in addition to other electrical remodeling of the SAN, reduced IKACh also plays an important role in the pregnancy-induced increased HR.

Similarities and differences in morphology, CD31 and CD68 expression of male vs. female sinoatrial node and its surrounding atrial muscle in ageing and obesity

Research purposeThe sinoatrial node (SN) is the main pacemaker site, and it is located in the junctional area of the superior vena cava within the right atrium (RA). The precise micro-anatomy of the SN in males and females in ageing and obesity remains unclear. Basic proceduresHuman SN/RA specimens were dissected from 25 post-mortem hearts (preserved in 4 % formaldehyde solution), under Polish local ethical rules. The SN/RA tissue blocks were embedded in paraffin. Masson's Trichrome staining and immunohistochemistry for CD31 (a marker of endothelial cells) and CD68 (a marker of macrophages) were performed. Images at different magnifications were taken and analysed. 12-lead ECGs from 24 patients under Polish local ethical rules were obtained. Heart rate and P wave morphologies from lead II, lead III and lead aVF were analysed. Statistical analysis was performed with the unpaired t-test. Principal resultsHeart weight to body weight ratio was significantly higher in aged obese males vs. their female counterparts. In the RA samples, there was an increase in connective tissue and decreased myocyte content from aged obese females compared to aged obese males. Aged non-obese males had significantly increased cellular hypertrophy than the aged non-obese females. Both the aged obese and aged non-obese females showed more CD3 but less CD68 expressing cells than males. In the SN samples, CD31 and CD68 expressing cells were higher in both aged non-obese and aged obese males than their female counterparts. Major conclusionsAgeing and/or obesity are more likely to impact these cardiac tissues through increased inflammation, increased immune response and hypertrophy.

Revealing alterations in heart rate fluctuations during the progression of Chagas disease.

The heart rate variability (HRV) continually evolves throughout life, reflecting modifications in the architecture of the sinoatrial node (SAN) and in the regulation of heart rate by the autonomic nervous system (ANS). Both can be considerably affected by Chagas disease, causing important changes in the complex nature of HRV. We aim to evaluate the ability of an index based on the false nearest neighbors method (FN10) to reflect these changes during disease progression. We perform a retrospective, descriptive, and cross-sectional study analyzing HRV time series of participants with Chagas disease. We determine the dependence of FN10 on age and sex in a healthy population, and then evaluate FN10 in individuals with Chagas disease. In the healthy population, FN10 has a scaling behavior with age, which is independent of sex. In Chagas disease, some individuals show FN10 values significantly above those seen in the healthy population. We relate the findings to the pathophysiological mechanisms that determine the progression of the disease. The results indicate that FN10 may be a candidate prognostic biomarker for heart disease.

Sex-Specific Differences in Cardiovascular Adaptations and Risks in Elite Athletes: Bridging the Gap in Sports Cardiology.

The growing participation of women in competitive sports necessitates a comprehensive understanding of sex-specific cardiovascular adaptations and risks. Historically, research has predominantly focused on male athletes, leaving a gap in knowledge about the unique cardiovascular dynamics of female peers. we hypothesized that female athletes exhibit distinct cardiovascular adaptations and face different risks, influenced by physiological, hormonal, and structural differences. A systematic review of the literature was conducted, analyzing studies on cardiovascular responses and adaptations in athletes. Data were extracted on hemodynamic changes, autonomic and neural reflex regulation, cardiac remodeling, and arrhythmias. Comparative analyses were performed to identify sex-specific patterns and discrepancies in cardiovascular health outcomes. We revealed considerable sex differences in cardiovascular adaptations to athletic training. Female athletes generally have longer QT intervals, greater sinoatrial node automaticity, and enhanced atrioventricular node function compared to males. They also exhibit lower sympathetic activity, lower maximal stroke volumes, and a tendency toward eccentric cardiac remodeling. Conversely, male athletes are more prone to concentric hypertrophy and higher incidences of bradyarrhythmia and accessory pathway arrhythmias. Female athletes are more likely to experience symptomatic atrial fibrillation and face higher procedural complications during catheter ablation. Our findings underscore the necessity for sex-specific approaches in sports cardiology. Recognizing and addressing these differences could enhance performance and reduce adverse cardiac events in athletes. Future research should focus on developing tailored screening, prevention, and treatment strategies to bridge the knowledge gap and promote cardiovascular health in both male and female athletes.

Sigmoidal heart rate response pattern during exercise in patients with chronic heart failure.

Detailed heart rate (HR) response patterns during exercise in patients with heart failure (HF) and sinus rhythm remain uncertain. We screened consecutive patients with HF who underwent cardiopulmonary exercise tests at a large academic center from November 2013 to July 2023. HR response during exercise was statistically classified using logistic differential equation models. A total of 99 patients were included. Of them, 75 patients were assigned to "sigmoidal pattern" and the other 24 to "exponential pattern." Patients with the sigmoidal pattern were older and exhibited higher plasma B-type natriuretic peptide levels. Increases in HR and oxygen consumption (V̇o2)/kg up to the anaerobic threshold level were not different between both patterns. However, beyond the threshold, the sigmoidal pattern group showed no further increase in HR and significantly lower V̇o2/kg than their counterparts (interactions for P < 0.001). HR response during exercise in patients with heart failure and sinus rhythm was categorized into two unique groups: sigmoidal and exponential patterns. More detailed clarification of the sigmoidal pattern, potentially indicating sinus node dysfunction, should offer new clinical insights for chronotropic incompetence.NEW & NOTEWORTHY Heart rate response patterns can be classified into two groups among patients with chronic heart failure reaching maximal exertion: sigmoidal and exponential.

Children and Adolescent Patients with Variants in the ATP1A3 -encoded Sodium-Potassium ATPase Alpha-3 Subunit Demonstrate an Impaired QT Response to Bradycardia and Predisposition to Sinus Node Dysfunction.

Alternating hemiplegia of childhood (AHC) is a rare disorder with both neurologic and cardiac manifestations. The ATP1A3-D801N variant is associated with a pathologically short QT interval and risk of ventricular arrhythmia following bradycardia; however, the mechanism of this remains unknown. We investigated the relationship between heart rate (HR), QT, and QTc, hypothesizing that individuals with ATP1A3-D801N have abnormal, impaired shortening of QT and QTc at lower HR leading to arrhythmia predisposition. We performed a retrospective observational study of individuals who underwent clinical evaluation, Holter monitoring, and genetic testing for AHC at Duke University Hospitals. We also compiled a group of healthy individuals as a control cohort. A larger, worldwide cohort of individuals with ATP1A3 - related phenotypes was compiled to investigate sinus node dysfunction. Linear regression analysis was then performed. The cohort consisted of 44 individuals with ATP1A3 -related phenotypes with 81 Holter recordings (52.27% female; mean age at first Holter 8.04 years, range 0.58 - 33 years), compared to 36 healthy individuals with 57 Holter recordings (52.78% female; mean age at first Holter 9.84 years, range 0.08 - 38 years). Individuals with ATP1A3-D801N had reduced prolongation of QT at lower HR, manifest as a significantly lower slope for HR vs QT compared to healthy (P<0.0001). This resulted in a significantly higher slope of the relationship for HR vs QTc compared to healthy (P<0.0001). Individuals with ATP1A3 - related phenotypes and baseline QTc <350 milliseconds (ms) had increased shortening of QT and QTc at lower HR compared to those with normal QTc (P=0.003; P=0.001). Among worldwide cases, 3 out of 131 individuals with ATP1A3 -related phenotypes required device implantation and/or had sinus pauses >4 seconds. Individuals with the ATP1A3-D801N variant exhibit paradoxical shortening of QT and QTc at lower HR, which contribute to an increased risk of arrhythmias during bradycardia. This is exacerbated by an underlying risk of sinus node dysfunction. What is Known: Individuals with ATP1A3-D801N have a short baseline QTc.Two individuals with AHC experienced ventricular fibrillation following bradycardia. What the Study Adds: The QT and QTc shorten to a greater extent at lower heart rate in individuals with ATP1A3-D801N than in healthy individuals. Individuals with ATP1A3 -related phenotypes and QTc <350ms show greater impairment of QT and QTc dynamics than those with normal QTc. There is low prevalence of device implantation and significant sinus pauses in individuals with ATP1A3 -related phenotypes, with a relatively greater prevalence in those with ATP1A3-D801N.

Modulation of Spontaneous Action Potential Rate by Inositol Trisphosphate in Myocytes from the Rabbit Atrioventricular Node.

The atrioventricular node (AVN) is a key component of the cardiac conduction system and takes over pacemaking of the ventricles if the sinoatrial node fails. IP3 (inositol 1,4,5 trisphosphate) can modulate excitability of myocytes from other regions of the heart, but it is not known whether IP3 receptor (IP3-R) activation modulates AVN cell pacemaking. Consequently, this study investigated effects of IP3 on spontaneous action potentials (APs) from AVN cells isolated from rabbit hearts. Immunohistochemistry and confocal imaging demonstrated the presence of IP3-R2 in isolated AVN cells, with partial overlap with RyR2 ryanodine receptors seen in co-labelling experiments. In whole-cell recordings at physiological temperature, application of 10 µM membrane-permeant Bt3-(1,4,5)IP3-AM accelerated spontaneous AP rate and increased diastolic depolarization rate, without direct effects on ICa,L, IKr, If or INCX. By contrast, application via the patch pipette of 5 µM of the IP3-R inhibitor xestospongin C led to a slowing in spontaneous AP rate and prevented 10 µM Bt3-(1,4,5)IP3-AM application from increasing the AP rate. UV excitation of AVN cells loaded with caged-IP3 led to an acceleration in AP rate, the magnitude of which increased with the extent of UV excitation. 2-APB slowed spontaneous AP rate, consistent with a role for constitutive IP3-R activity; however, it was also found to inhibit ICa,L and IKr, confounding its use for studying IP3-R. Under AP voltage clamp, UV excitation of AVN cells loaded with caged IP3 activated an inward current during diastolic depolarization. Collectively, these results demonstrate that IP3 can modulate AVN cell pacemaking rate.

Simulation of predicting atrial fibrosis in patients with paroxysmal atrial fibrillation during sinus node recovery time in optical imaging

Paroxysmal atrial fibrillation is a common arrhythmia, and its development process and prediction of the degree of atrial fibrosis are of great significance for treatment and management. Optical imaging technology provides a new means for non-invasive observation of atrial electrical activity. The aim of this study is to investigate the predictive effect of sinus node recovery time on the degree of atrial fibrosis in patients with paroxysmal atrial fibrillation, and to provide a basis for the application of optical imaging technology in the study of atrial fibrosis. The study collected clinical and optical imaging data from a group of patients with paroxysmal atrial fibrillation, and used statistical analysis methods to investigate the relationship between sinus node recovery time and the degree of atrial fibrosis. The research results indicate that there is a significant correlation between the recovery time of the sinus node and the degree of atrial fibrosis, that is, there is a positive correlation between the prolonged recovery time of the sinus node and the aggravation of atrial fibrosis. SNRT can serve as an effective indicator for evaluating atrial matrix and can be applied to predict recurrence after catheter ablation of paroxysmal atrial fibrillation. Shortening SNRT through catheter ablation can become an important predictor of effective catheter ablation.

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.

Navigating Complexity in Postural Orthostatic Tachycardia Syndrome.

Postural Orthostatic Tachycardia Syndrome (POTS) affects up to 1% of the US population, predominantly women, and is characterized by a complex, elusive etiology and heterogeneous phenotypes. This review delves into the intricate physiology and etiology of POTS, decoding the roles of the sinoatrial node, the autonomic nervous system, fluid dynamics, and the interplay between the immune and endocrine systems. It further examines key contributing factors such as dysautonomia, thoracic hypovolemia, autonomic neuropathies, sympathetic denervation, autoimmune responses, and associations with conditions such as small-fiber neuropathy and mast cell activation syndrome. Given the numerous mysteries surrounding POTS, we also cautiously bring attention to sinoatrial node and myocardial function, particularly in how the heart responds to stress despite exhibiting a normal cardiac phenotype at rest. The potential of genomic research in elucidating the underlying mechanisms of POTS is emphasized, suggesting this as a valuable approach that is likely to improve our understanding of the genetic underpinnings of POTS. The review introduces a tentative classification system for the etiological factors in POTS, which seeks to capture the condition's diverse aspects by categorizing various etiological factors and acknowledging co-occurring conditions. This classification, while aiming to enhance understanding and optimize treatment targets, is presented as a preliminary model needing further study and refinement. This review underscores the ongoing need for research to unravel the complexities of POTS and to develop targeted therapies that can improve patient outcomes.

Role of new generation implantable loop recorders in managing undiagnosed pediatric cardiac symptoms

Pediatric cardiac symptoms such as palpitations, syncope, or seizure-like episodes pose diagnostic challenges for general pediatricians. These symptoms, though often benign, may reveal underlying arrhythmias or inherited cardiac conditions (ICCs), affecting the quality of life and limiting activity participation. The purpose of this study is to determine the effectiveness and safety of implantable loop recorders (ILRs) in diagnosing and managing arrhythmias in pediatric patients. A retrospective cohort study conducted over an 8-year period from January 2016 to December 2023 in a single pediatric cardiology center. A cohort of 155 pediatric patients (median age 11.4 years) who underwent ILR implantation were selected based on symptoms such as palpitations, chest pain, or syncope, and those with previously recorded arrhythmias or high-risk ICCs. The primary outcomes were the diagnostic yield of ILRs for arrhythmias and subsequent changes in patient management. Diagnostic yield was defined as the detection of relevant arrhythmias, such as pauses of 3 s or longer, high-degree AV block, sinus node dysfunction, supraventricular tachycardia, ventricular tachycardia, or inappropriate sinus tachycardia. The median follow-up period was 2.3 years (845 days). Diagnostic arrhythmias were recorded in 60% of patients with symptom-activated transmissions and 80% of device-activated transmissions. Sinus pauses (37.5%) and VT (30%) were the most common arrhythmias detected. In patients with syncope (n = 76), 30% had relevant arrhythmias. In the palpitations group (n = 20), 35% had relevant arrhythmias. Approximately 80% of patients with ILR-diagnosed arrhythmias underwent targeted management, including medication changes and additional procedures. No significant complications were observed; minor complications occurred in 2.5% of patients.Conclusions: New generation ILRs are effective and safe for diagnosing and managing pediatric arrhythmias, providing significant reassurance to patients and families. Further studies are needed to evaluate the impact of ILRs on quality of life and sports participation in high-risk young patients.What is Known:• Pediatric Cardiac Symptoms: Symptoms like palpitations, syncope, and seizure-like episodes in pediatric patients pose significant diagnostic challenges.• Diagnostic Tools: Traditional diagnostic tools such as ECG, Holter monitors, and external loop recorders often have low diagnostic yields due to the sporadic nature of symptoms.• Implantable Loop Recorders (ILRs): ILRs have been shown to improve the diagnostic yield for arrhythmias in adults and provide long-term rhythm monitoring.What is New:• Effectiveness in Pediatrics: This study demonstrates that new generation ILRs significantly enhance the diagnostic yield for arrhythmias in pediatric patients with undiagnosed cardiac symptoms.• Safety and Efficacy: The study confirms that ILRs are both safe and effective in the pediatric population, with minimal complications and high diagnostic accuracy.• Targeted Management: Findings show that ILR-diagnosed arrhythmias lead to targeted management changes in approximately 80% of cases, including medication adjustments and additional procedures.• Long-term Monitoring: ILRs provide continuous long-term monitoring, offering reassurance to patients and families, and potentially improving quality of life and participation in activities for high-risk young patients.

Kv1.1 channels help set the pace.

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.

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.

Abstract Mo079: Analyzing the Predicted Impact of Published SCN5A Variants in Juvenile-Onset Sick Sinus Syndrome

Background: Sick sinus syndrome (SSS) is a cardiac condition caused by sinoatrial node dysfunction, leading to arrhythmias due to the inability to transmit signals properly to the upper chambers of the heart. While age-related degeneration of the sinoatrial node is the most common contributing factor to SSS, congenital disorders involving genetic variants can also lead to its development, even in pediatric patients. To explore the genetic basis of SSS in the pediatric population (age &lt; 19 years), we conducted a comprehensive review of case reports to identify associated genetic variants. Our focus centered on variants reported in the sodium channel protein type 5 subunit alpha ( SCN5A ) gene, which has been reported to contribute to various ventricular arrhythmias, including SSS. Methods: A comprehensive literature review of case reports of juvenile-onset SSS was conducted using PubTator/PubMed and the Online Mendelian Inheritance in Man (OMIM) database to identify SCN5A variants. Case reports were selected if they met the following criteria: at least one patient diagnosed with SSS at or under the age of 19 with a detected variant in the SCN5A gene. The structural effects of identified variants were assessed using the HOPE web service. Results: Data compiled from the literature review provided a total of 53 cases related to juvenile-onset SSS and the SCN5A gene. Among the reported patients, many exhibited multiple arrhythmogenic phenotypes alongside SSS, including progressive cardiac conduction disease, idiopathic ventricular tachycardia, and Brugada syndrome. The majority of identified variants were missense, although truncating and insertion/deletion variants were also documented. All three inheritance variations - heterozygous, homozygous, and compound heterozygous - were observed in this set of patients. Although many of the variants had some functional studies performed to determine their pathogenicity, some of the variants did not have a predicted impact. For these variants, we characterized the location and biochemical properties of the variants, comparing them to the variants with known functional impact. Conclusion: Future studies will explore the functional impact associated with the predicted variants through modeling specific mutations in SCN5A , using cardiomyocytes in vitro . Additionally, using further published studies and surveying the protein structure analysis of SCN5A variants will aid in characterizing those that are specific to SSS.

Abstract Mo072: Sustained Biological Pacemaker Activity Induced by AAV9-hTBX18

Background: Sinoatrial node (SAN) dysfunction is a clinical disorder characterized by the inability of the SAN to maintain an appropriate heart rate (HR). Patients with this condition often need an electronic pacemaker. Although it is the current standard of care, devices can have complications. A biological pacemaker has been created by adenovirus expressing TBX18 as an alternative. Hypothesis: However, adenoviruses are immunogenic, limiting the duration of expression. We hypothesized that an AAV9 would promote prolonged TBX18 expression, induce somatic reprogramming in vivo, and sustained pacemaker activity. Methods: AAV9 containing GFP or TBX18 was directly injected into the myocardium of eight-week-old male Sprague Dawley rats (n=70) at low (1x10 9 GC), mid (1x10 10 GC) and high (1x10 11 GC) doses. Transient AV block was induced by tail vein injection of adenosine. A permanent complete AV block was induced by ablation of the his bundle. Hearts were harvested for optical mapping and mRNA analysis. Results: Six weeks after injection, all doses of AAV9-TBX18 had increased HR (low: 104±4, p=0.04; mid: 106±3.4, p=0.008; high: 103±4.1, p=0.04; vs GFP: 89±3 bpm) compared to AAV9-GFP when we induced AV block using adenosine. AAV9-GFP and -TBX18 mid dose injected rats underwent surgery to induce complete AV block. TBX18 group presented superior HR over the 24 hours monitored. 6-lead EKG analysis of the QRS axis in the frontal plane revealed that the ventricular ectopic beats from the AAV9-TBX18-injected group originated from the injection site. Activation maps further confirmed that the injection site originated those beats in TBX18-injected rats only. Successful reprogramming of adult ventricular myocytes into SAN-like cells was confirmed by transcriptional profiles with increased expression of TBX18 (2.2±0.5 FC, p&lt;0.0001) and Hcn2 (25.2±1.1 FC, p=0.02), and reduced Gja1 (-10.8±0.7 FC, p=0.005), Scn5a (-1.1±0.8 FC, p=0.01) and Nkx2.5 (-2.0±0.6 FC, p=0.004) relative to GFP. Conclusion: Collectively, these data indicate that the AAV-based somatic reprogramming induced by TBX18 leads to sustained biological pacemaker activity in rats with AV block. Long-term studies are needed to characterize the long-term effects of sustained TBX18 expression.

Abstract We042: Gelatin hydrogel elasticity influences TBX18-induced pacemaker cell behavior

Introduction: Extracellular matrix (ECM) stiffness plays a role in determining cell behavior during development and maintenance of organ function. Polystyrene dishes typically show an elastic modulus of 1 gigaPa, which is far from the elastic modulus of 10 to 15 kiloPa for myocardium. Gelatin, a derivative of collagen, is a major structural scaffolding in the myocardium including the sinoatrial node. Hypothesis: We hypothesized that gelatin hydrogel at varying stiffness serves as a platform to modulate the automaticity of cardiac pacemaker cells. Methods: Freshly isolated neonatal rat ventricular myocytes (NRVMs) were cultured as monolayers on regular, plastic cell culture plate or on 3%, 5%, and 10% gelatin hydrogel that represents a stiffness of 1.3 ± 0.3, 6.4 ± 1.5, and 13.6 ± 3.5 kPa, respectively. All cell culture surface was coated with fibronectin. NRVMs were transduced with Adv-TBX18 or Adv-GFP at day 0 (d0). Results: TBX18-NRVMs showed significantly more spontaneous synchronous contractions on the gelatin hydrogel (22 ± 12, 29 ± 1, 33 ± 9 on 3, 5, or 10% gelatin, respectively) than on plastic (10 ± 11) at d4. Control, GFP-NRVMs showed few spontaneous synchronous contractions regardless of the cell culture platform (3 ± 4, 2 ± 3, 0 ± 0 bpm at 3, 5, 10% gelatin, respectively; 3 ± 7 bpm on plastic, n = 5 to 6 wells/group) at d4. TBX18-NRVMs exhibited significantly more spontaneous synchronous Ca 2+ transients/min on 10% gelatin hydrogel (33 ± 2) than on 3% gelatin hydrogel (9 ± 2) or 5% gelatin hydrogel (19 ± 8) (n = 3, p&lt;0.05) at d8. Immunostaining revealed that Hcn4 protein expression in TBX18-NRVMs was higher on 10% gelatin than on plastic at d2. The percentage of vimentin+ nonmyocytes in TBX18-NRVMs was higher on 10% gelatin (39.5 ± 2.2 %) compared to either on 5% (26.6 ± 1.7%) or on 3% gelatin (27.5 ± 4.2) at d6. Conclusions: Our data indicate that spontaneous and synchronized contractions of TBX18-induced pacemaker cells were significantly higher on gelatin hydrogel with myocardium-like stiffness compared to those grown on plastic culture medium. The data suggest that modulating ECM stiffness may impact the automaticity of cardiac pacemaker tissues, and could serve as a platform to study sinus node dysfunction.

Transcriptional regulation of the postnatal cardiac conduction system heterogeneity

The cardiac conduction system (CCS) is a network of specialized cardiomyocytes that coordinates electrical impulse generation and propagation for synchronized heart contractions. Although the components of the CCS, including the sinoatrial node, atrioventricular node, His bundle, bundle branches, and Purkinje fibers, were anatomically discovered more than 100 years ago, their molecular constituents and regulatory mechanisms remain incompletely understood. Here, we demonstrate the transcriptomic landscape of the postnatal mouse CCS at a single-cell resolution with spatial information. Integration of single-cell and spatial transcriptomics uncover region-specific markers and zonation patterns of expression. Network inference shows heterogeneous gene regulatory networks across the CCS. Notably, region-specific gene regulation is recapitulated in vitro using neonatal mouse atrial and ventricular myocytes overexpressing CCS-specific transcription factors, Tbx3 and/or Irx3. This finding is supported by ATAC-seq of different CCS regions, Tbx3 ChIP-seq, and Irx motifs. Overall, this study provides comprehensive molecular profiles of the postnatal CCS and elucidates gene regulatory mechanisms contributing to its heterogeneity.