Sinoatrial Node Preparations Research Articles

PO-01-172 METABOLIC SHIFT CONTRIBUTES TO SINOATRIAL NODE DYSFUNCTION IN HEART FAILURE WITH PRESERVED EJECTION FRACTION

Cardiometabolic alterations are frequent in patients with heart failure with preserved ejection fraction (HFpEF). We recently demonstrated sinoatrial node (SAN) dysfunction limits the chronotropic response in two distinct animal models of HFpEF; however, our new findings suggest a relationship between metabolism and SAN dysfunction. To identify metabolically-driven changes that facilitate SAN dysfunction in HFpEF. Male C57Bl6 mice fed with high fat (HFD) plus nitric oxide inhibitor (L-NAME) diet or regular chow served as HFpEF and controls, respectively. Transcriptomics, metabolomics, targeted quantitative proteomics of mitochondria, and optical mapping of SAN preparations from control and HFpEF-verified mice were performed. SAN from HFpEF-verified animals after 20 weeks of HFD+LNAME exhibited prolonged SAN recovery time (100%; P<0.05). A combination of multi-omics studies revealed changes in metabolic gene clusters suggesting a metabolic shift towards glycolysis as a source of energy. Supporting these findings, isolated mitochondria from HFpEF SANs showed depressed maximal mitochondrial respiration compared to controls (-42%; P<0.05). In addition, isolated pacemaker cells from HFpEF animals showed increased mitochondrial reactive oxygen species production compared to controls (+70%; P<0.05). Moreover, acute mitochondrial uncoupling induced by FCCP elicited a pronounced prolongation of SAN recovery time in HFpEF compared to controls (165%; P<0.05). Our results suggest that SAN dysfunction in HFpEF is closely associated with metabolic remodeling and energetic substrate shift. However, further studies are needed to establish the causality between metabolic changes and SAN dysfunction in HFpEF.

Autonomic innervation, a peripheral glial cell web, and a novel S100B expressing interstitial cell type impart structural and functional complexity to the sinoatrial node.

The sinoatrial node (SAN) of the heart produces rhythmic action potentials, generated via calcium signaling within and among pacemaker cells. Our previous work has described the SAN as composed of a HCN4+-pacemaker cell meshwork, which merges with a network of connexin 43+/F-actin+ cells. It is also known that sympathetic and parasympathetic innervation create an autonomic plexus in the SAN that modulates heart rate and rhythm. However, the anatomical details of the interaction of this plexus with the pacemaker cell meshwork have yet to be described. The cytoarchitecture of SAN whole-mount preparations was examined by three-dimensional confocal laser scanning microscopy of triple immunolabeled with combinations of antibodies for HCN4, S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP), choline acetyltransferase, or vesicular acetylcholine transporter, and tyrosine hydroxylase, and transmission electron microscopy. The SAN exhibited heterogeneous autonomic innervation, which was accompanied by a web of peripheral glial cells and a novel S100B+/GFAP- interstitial cell population, with a unique morphology and a distinct distribution pattern, creating complex interactions with other cell types in the node, particularly with HCN4-expressing cells. Transmission electron microscopy identified a similar population of interstitial cells as telocytes, which appeared to secrete vesicles toward pacemaker cells. Application of S100B to SAN preparations desynchronized Ca2þ signaling in HCN4-expressing cells and increased variability in SAN impulse rate and rhythm. The autonomic plexus, peripheral glial cell web, and a novel S100Bþ/GFAP- interstitial cell type embedded within the HCN4þ cell meshwork increase the structural and functional complexity of the SAN and provide a new regulatory pathway of rhythmogenesis.

The Heart's Pacemaker Mimics Brain Cytoarchitecture and Function: Novel Interstitial Cells Expose Complexity of the SAN.

The sinoatrial node (SAN) of the heart produces rhythmic action potentials, generated via calcium signaling within and among pacemaker cells. Our previous work has described the SAN as composed of a hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4)-expressing pacemaker cell meshwork, which merges with a network of connexin 43⁺/F-actin⁺ cells. It is also known that sympathetic and parasympathetic innervation create an autonomic plexus in the SAN that modulates heart rate and rhythm. However, the anatomical details of the interaction of this plexus with the pacemaker cell meshwork have yet to be described. This study sought to describe the 3-dimensional cytoarchitecture of the mouse SAN, including autonomic innervation, peripheral glial cells, and pacemaker cells. The cytoarchitecture of SAN whole-mount preparations was examined by three-dimensional confocal laser-scanning microscopy of triple immunolabeled with combinations of antibodies for HCN4, S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP), choline acetyltransferase, or vesicular acetylcholine transporter, and tyrosine hydroxylase, and transmission electron microscopy. The SAN exhibited heterogeneous autonomic innervation, which was accompanied by a web of peripheral glial cells and a novel S100B⁺/GFAP^- interstitial cell population, with a unique morphology and a distinct distribution pattern, creating complex interactions with other cell types in the node, particularly with HCN4-expressing cells. Transmission electron microscopy identified a similar population of interstitial cells as telocytes, which appeared to secrete vesicles toward pacemaker cells. Application of S100B to SAN preparations desynchronized Ca²⁺ signaling in HCN4-expressing cells and increased variability in SAN impulse rate and rhythm. The autonomic plexus, peripheral glial cell web, and a novel S100B⁺/GFAP^- interstitial cell type embedded within the HCN4⁺ cell meshwork increase the structural and functional complexity of the SAN and provide a new regulatory pathway of rhythmogenesis.

Role of Endothelin-1 in Right Atrial Arrhythmogenesis in Rabbits with Monocrotaline-Induced Pulmonary Arterial Hypertension.

Atrial arrhythmias are considered prominent phenomena in pulmonary arterial hypertension (PAH) resulting from atrial electrical and structural remodeling. Endothelin (ET)-1 levels correlate with PAH severity and are associated with atrial remodeling and arrhythmia. In this study, hemodynamic measurement, western blot analysis, and histopathology were performed in the control and monocrotaline (MCT, 60 mg/kg)-induced PAH rabbits. Conventional microelectrodes were used to simultaneously record the electrical activity in the isolated sinoatrial node (SAN) and right atrium (RA) tissue preparations before and after ET-1 (10 nM) or BQ-485 (an ET-A receptor antagonist, 100 nM) perfusion. MCT-treated rabbits showed an increased relative wall thickness in the pulmonary arterioles, mean cell width, cross-sectional area of RV myocytes, and higher right ventricular systolic pressure, which were deemed to have PAH. Compared to the control, the spontaneous beating rate of SAN–RA preparations was faster in the MCT-induced PAH group, which can be slowed down by ET-1. MCT-induced PAH rabbits had a higher incidence of sinoatrial conduction blocks, and ET-1 can induce atrial premature beats or short runs of intra-atrial reentrant tachycardia. BQ 485 administration can mitigate ET-1-induced RA arrhythmogenesis in MCT-induced PAH. The RA specimens from MCT-induced PAH rabbits had a smaller connexin 43 and larger ROCK1 and phosphorylated Akt than the control, and similar PKG and Akt to the control. In conclusion, ET-1 acts as a trigger factor to interact with the arrhythmogenic substrate to initiate and maintain atrial arrhythmias in PAH. ET-1/ET-A receptor/ROCK signaling may be a target for therapeutic interventions to treat PAH-induced atrial arrhythmias.

EN-510-03 SINOATRIAL NODE DYSFUNCTION IN HEART FAILURE WITH PRESERVED EJECTION FRACTION

Under normal conditions, the human heart beats over 2 billion times during an average lifetime due to rhythmic electrical impulses initiated in the sinoatrial node (SAN). In disease conditions, such as heart failure with preserved ejection fraction (HFpEF), failure to maintain and regulate the heart rate due to SAN dysfunction can occur. We recently identified latent SAN dysfunction in animal models of cardiometabolic HFpEF. However, little is known about the metabolic imbalances underlying SAN dysfunction in HFpEF. To identify metabolically-driven functional and transcriptomic changes in HFpEF SAN. Male C57Bl6 mice fed with high fat (HFD) plus nitric oxide inhibitor (L-NAME) diet or regular chow served as HFpEF and control, respectively. RNA sequencing and optical mapping of SAN preparations from control and HFpEF-verified mice were performed. After 20 weeks of HFD+L-NAME, the sinus node recovery time was significantly prolonged in HFpEF mice compared to controls (162 ± 25 vs 88 ± 19 ms; p<0.05). Transcriptome profiling of SAN tissue revealed a significant enhancement of multiple disease-associated genes, with striking changes in extracellular matrix genes and metabolic pathways. In concordance with the observed fibrotic remodeling, conduction velocity (CV) was significantly lower in SAN of HFpEF mice compared to controls (6.4 ± 0.5 vs 13.1 ± 1.7 cm/s; p<0.05). Moreover, although β-adrenergic receptor stimulation accelerated the CV in control SAN, failed to do so in HFpEF animals (20 ± 3.1 vs 10.2 ± 1.1 cm/s; p<0.05). Our results using a cardiometabolic HFpEF model indicate that SAN dysfunction is closely associated with molecular changes in metabolic pathways and extracellular matrix remodeling associated genes. Understanding the metabolic control of the SAN may open new therapeutic targets for HFpEF associated SAN dysfunction.

Mama and papa of the early spontaneous electrical activity of the embryo sinoatrial node

In adult mammalian hearts the spontaneous diastolic depolarization of the sinoatrial node (SAN) cells is generated by two interacting systems, a so‐called “membrane‐clock” and “calcium‐clock”. Hyperpolarization‐activated pacemaker current (If) plays a key role in the “membrane‐clock”, while the “calcium‐clock” is based on rhythmic spontaneous Ca2+ release from sarcoplasmic reticulum via ryanodine receptors (RyRs). These complementing systems provide regular pacemaking even in case of damage of one of them. The aim of this study was to evaluate a role of If current and ryanodine receptors in the electrical activity of the developing mouse SAN.MethodsWe used embryos of mice, aged of E9.5, E12.5 and newborn to perform our experiments. After release from the amniotic membrane, the embryos were transferred with a pipette to a clean Petri dish with oxygenated physiological solution containing (in mM/L) 140 NaCl, 5.4 KCl, 0.1 CaCl2, 0.33 NaH2PO4, 1 MgCl2, 10 HEPES, 10 Glucose, (pH 7.38) at room temperature. To record spontaneous action potential (AP) we used conventional microelectodes technique at 37◦C. Differences were considered significant at p &lt; 0.05ResultsMicroelectrode mapping at E9.5 revealed that the SAN cells are located in the inflow tract of the venous pole. At E12 the SAN cells are located in the cavo‐auricular junction. In neonatal mice, SAN cells were detected at intercaval area near to the crista terminalis. At E9.5, in 6 experiments, on 25‐ 30 minute exposure of 3 mkM ivabradine (Iva) an irregularity in AP generation by SAN cells was occurred. The same effects were obtained in 4 experiments on 20‐30 minutes exposure of 3 mkM ryanodine. At E12.5 exposure of Iva 3 mkM for 30 minutes reduced the pacemaker firing rate by 34% (p &lt; 0.05) but did not break the rhythmic generation. In contrast, the exposure of 3 mkM ryanodine for 30 minutes, in 4 experiments, still caused an irregularity in AP generation by SAN cells. In neonatal isolated SAN preparations, exposure of Iva 3 mkM and 3 mkM ryanodine for 30 minutes reduced the pacemaker firing rate equally (on 32%; p &lt; 0.05). However, co‐application of Iva and ryanodine induced an irregularity in AP generation by SAN cells (Figure).Numerical experiments within the framework of mouse SANC model (Kharche, AJP, 11) adopted for embryonic cells (Markov, IEEE, 21) have shown the decrease in the frequency of the AP generation with decreasing of the “funny” currents permeability caused by Iva action.The supposed complete RyRs opening, caused by a relatively low ryanodine concentration, led to a decrease of the AP generation frequency, but not to the rhythm disturbances. However a decrease in RyRs permeability by an order of magnitude led to the appearance of “bursts‐like” irregularity in AP generation.ConclusionThus, the membrane‐clock and the Ca2+‐clock mechanisms is critical for spontaneous electrical activity E9.5 mouse embryos, whereas the Ca2+‐clock mechanisms is critical for increase rate AP generation in development SAN cells.

Natriuretic peptide receptor B maintains heart rate and sinoatrial node function via cyclic GMP-mediated signalling.

Heart rate (HR) is a critical indicator of cardiac performance that is determined by sinoatrial node (SAN) function and regulation. Natriuretic peptides, including C-type NP (CNP), have been shown to modulate ion channel function in the SAN when applied exogenously. CNP is the only NP that acts as a ligand for natriuretic peptide receptor-B (NPR-B). Despite these properties, the ability of CNP and NPR-B to regulate HR and intrinsic SAN automaticity in vivo, and the mechanisms by which it does so, are incompletely understood. Thus, the objective of this study was to determine the role of NPR-B signalling in regulating HR and SAN function. We have used NPR-B deficient mice (NPR-B+/-) to study HR regulation and SAN function using telemetry in conscious mice, intracardiac electrophysiology in anaesthetized mice, high-resolution optical mapping in isolated SAN preparations, patch-clamping in isolated SAN myocytes, and molecular biology in isolated SAN tissue. These studies demonstrate that NPR-B+/- mice exhibit slow HR, increased corrected SAN recovery time, and slowed SAN conduction. Spontaneous AP firing frequency in isolated SAN myocytes was impaired in NPR-B+/- mice due to reductions in the hyperpolarization activated current (If) and L-type Ca2+ current (ICa,L). If and ICa,L were reduced due to lower cGMP levels and increased hydrolysis of cAMP by phosphodiesterase 3 (PDE3) in the SAN. Inhibiting PDE3 or restoring cGMP signalling via application of 8-Br-cGMP abolished the reductions in cAMP, AP firing, If, and ICa,L, and normalized SAN conduction, in the SAN in NPR-B+/- mice. NPR-B+/- mice did not exhibit changes in SAN fibrosis and showed no evidence of cardiac hypertrophy or changes in ventricular function. NPR-B plays an essential physiological role in maintaining normal HR and SAN function by modulating ion channel function in SAN myocytes via a cGMP/PDE3/cAMP signalling mechanism.

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice.

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

Conserved Role of the Large Conductance Calcium-Activated Potassium Channel, KCa1.1, in Sinus Node Function and Arrhythmia Risk.

KCNMA1 encodes the α-subunit of the large-conductance Ca2+-activated K+ channel, KCa1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of KCa1.1 are limited, and KCNMA1 has not been investigated as an AF candidate gene. The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of KCa1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. A complex KCNMA1 variant was identified in 1 kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of KCa1.1 in normal hearts using immunostaining and immunogold electron microscopy. KCa1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the KCa1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the KCa1.1 ortholog, kcnma1b, in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila KCa1.1 ortholog, slo, systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo-deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the KCa1.1 loss-of-function models. Our data point to a highly conserved role of KCa1.1 in sinus node function in humans, mice, zebrafish, and fly and suggest that KCa1.1 loss of function may predispose to AF.

Trastuzumab increases pulmonary vein arrhythmogenesis through modulating pulmonary vein electrical and conduction properties via phosphatidylinositol 3-kinase signaling.

Objective(s):Drug-induced atrial fibrillation (AF) is considered an adverse effect of chemotherapeutic drugs. AF is a crucial risk factor for stroke, heart failure, myocardial infarction, and mortality. Pulmonary veins (PVs) are considered triggers inducing AF, and the sinoatrial node (SAN) may modulate PV activity and participate in AF genesis. AF was associated with early discontinuation of trastuzumab in patients with breast cancer. However, whether trastuzumab directly modulates the electrophysiological characteristics of PV and SAN remains unclear. Materials and Methods:ECG and conventional microelectrode system were used to record rabbit heart rhythm in vivo and electrical activities in vitro from isolated SAN, PV, and SAN-PV preparations. Results:Trastuzumab reduced the beating rate in isolated PV and SAN preparations at 1, 10, and 30 μM (particularly in isolated SAN preparations) and induced burst firings in isolated PV preparations at 10 μΜ. In addition, trastuzumab (10 μM) induced SAN-PV conduction block and burst firings, which were blocked by wortmannin (a PI3K inhibitor, 100 nM). Similarly, ECG recordings showed that acute intravenous administration of trastuzumab (10 mg/kg) reduced rabbit heart rates. Conclusion:Trastuzumab increased PV arrhythmogenesis through interfering with PI3K signaling, which may contribute to the genesis of AF.

Atrial arrhythmogenesis in a rabbit model of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) increases the risk of atrial fibrillation (AF), however, its arrhythmogenic mechanisms are unclear. This study investigated the effects of COPD on AF triggers (pulmonary veins, PVs) and substrates (atria), and their potential underlying mechanisms. Electrocardiographic, echocardiographic, and biochemical studies were conducted in control rabbits and rabbits with human leukocyte elastase (0.3 unit/kg)-induced COPD. Conventional microelectrode, Western blotting, and histological examinations were performed on PV, left atrium (LA), right atrium, and sinoatrial node (SAN) preparations from control rabbits and those with COPD. The rabbits with COPD had a higher incidence of atrial premature complexes, PV burst firing and delayed afterdepolarizations, higher sympathetic activity, larger LA, and faster PV spontaneous activity than did the control rabbits; but they exhibited a slower SAN beating rate. The LA of the rabbits with COPD had a shorter action potential duration and longer tachyarrhythmia induced by tachypacing (20 Hz) and isoproterenol (1 μM). Additionally, the rabbits with COPD had higher fibrosis in the PVs, LA, and SAN. H89 (10 μM), KN93 (1 μM), and KB-R7943 (10 μM) significantly suppressed burst firing and delayed afterdepolarizations in the PVs of the rabbits with COPD. Moreover, compared with the control rabbits, those with COPD had lower expression levels of the β1 adrenergic receptor, Cav 1.2, and Na+/Ca2+ exchanger in the PVs; Cav 1.2 in the LA; and hyperpolarization-activated cyclic nucleotide-gated K+ channel 4 in the SAN. COPD increases atrial arrhythmogenesis by modulating the distinctive electrophysiological characteristics of the PVs, LA, and SAN.

Aberrant sinus node firing during β-adrenergic stimulation leads to cardiac arrhythmias in diabetic mice.

Cardiovascular complications, including cardiac arrhythmias, result in high morbidity and mortality in patients with type-2 diabetes mellitus (T2DM). Clinical and experimental data suggest electrophysiological impairment of the natural pacemaker of the diabetic heart. The present study examined sinoatrial node (SAN) arrhythmias in a mouse model of T2DM and physiologically probed their underlying cause. Electrocardiogramswere obtained from conscious diabetic db/db and lean control db/+ mice. In vivo SAN function was probed through pharmacological autonomic modulation with isoprenaline, atropine and carbachol. Blood pressure stability and heart rate variability (HRV) were evaluated. Intrinsic SAN function was evaluated through ex vivo imaging of spontaneous Ca2+ transients in isolated SAN preparations. While lean control mice showed constant RR intervals during isoprenaline challenge, the diabetic mice experienced SAN arrhythmias with large RR fluctuations in a dose-dependent manner. These arrhythmias were completely abolished by atropine pre-treatment, while carbachol pretreatment significantly increased SAN arrhythmia frequency in the diabetic mice. Blood pressure and HRV were comparable in db/db and db/+ mice, suggesting that neither augmented baroreceptor feedback nor autonomic neuropathy is a likely arrhythmia mechanism. Cycle length response to isoprenaline was comparable in isolated SAN preparations from db/db and db/+ mice; however, Ca2+ spark frequency was significantly increased in db/db mice compared to db/+ at baseline and after isoprenaline. Our results demonstrate a dysfunction of cardiac pacemaking in an animal model of T2DM upon challenge with a β-adrenergic agonist. Ex vivo, higher Ca2+ spark frequency is present in diabetic mice, which may be directly linked to in vivo arrhythmias.

Electrical Conduction System Remodeling in Streptozotocin-Induced Diabetes Mellitus Rat Heart.

Cardiovascular complications are common in type 1 diabetes mellitus (TIDM) and there is an increased risk of arrhythmias as a result of dysfunction of the cardiac conduction system (CCS). We have previously shown that, in vivo, there is a decrease in the heart rate and prolongation of the QRS complex in streptozotocin-induced type 1 diabetic rats indicating dysfunction of the CCS. The aim of this study was to investigate the function of the ex vivo CCS and key proteins that are involved in pacemaker mechanisms in TIDM. RR interval, PR interval and QRS complex duration were significantly increased in diabetic rats. The beating rate of the isolated sinoatrial node (SAN) preparation was significantly decreased in diabetic rats. The funny current density and cell capacitance were significantly decreased in diabetic nodal cells. Western blot showed that proteins involved in the function of the CCS were significantly decreased in diabetic rats, namely: HCN4, Cav1.3, Cav3.1, Cx45, and NCX1 in the SAN; RyR2 and NCX1 in the atrioventricular junction and Cx40, Cx43, Cx45, and RyR2 in the Purkinje network. We conclude that there are complex functional and cellular changes in the CCS in TIDM. The changes in the proteins involved in the function of this electrical system are expected to adversely affect action potential generation and propagation, and these changes are likely to be arrhythmogenic.

Factor Xa inhibitors differently modulate electrical activities in pulmonary veins and the sinoatrial node

Factor Xa inhibitors reduce stroke in patients with atrial fibrillation. Pulmonary veins (PVs) and the sinoatrial node (SAN) are crucial for genesis of atrial fibrillation. However, the electrophysiological effects of factor Xa inhibitors (edoxaban and rivaroxaban) on PVs and the SAN remain unclear. Conventional microelectrodes were used to record the action potential in isolated rabbit PVs and SAN preparations before and after administration of edoxaban (0.1, 0.3, and 1 μM) or rivaroxaban (0.01, 0.03, 0.1, and 0.3 μM). A whole-cell patch-clamp was used to record the late sodium current (INa-late) in isolated single rabbit PV cardiomyocytes. Edoxaban significantly reduced PV spontaneous beating rates at 0.3 and 1 μM (N = 6 rabbits, P < 0.05), and reduced SAN beating rates at 1 μM (N = 6, P < 0.05). Similarly, rivaroxaban reduced PV spontaneous beating rates at 0.1 and 0.3 μM (N = 7, P < 0.05), and reduced SAN beating rates at 0.3 μM (N = 6, P < 0.05). However, neither edoxaban (1 μM) nor rivaroxaban (0.3 μM) reduced PV spontaneous beating rates in the presence of 1 μM BMS200261 (an inhibitor of protease-activated receptors type 1, PAR1 inhibitor) or 10 μM ranolazine (an inhibitor of late sodium current, INa-late inhibitor). Edoxaban (0.3 and 1 μM) and rivaroxaban (0.1 and 0.3 μM) respectively decreased the INa-late by 47%, 47%, 36%, and 49% (n = 9 PV cardiomyocytes from 5 rabbits, P < 0.05). In conclusion, Factor Xa inhibitors reduce PV spontaneous activities and may modulate occurrence of atrial fibrillation by inhibiting PAR1 and reducing the INa-late in PVs.

Heart automaticity in mice lacking L-type Cav1.3 and T-type Cav3.1 Ca2+ channels: Insights into the cardiac pacemaker mechanism

Sino-atrial node (SAN) pacemaker activity is generated by ion channels of the plasma membrane, such as hyperpolarization-activated “funny” f-(HCN), Ca 2+ channels and ryanodine receptor (RyR) – dependent Ca 2+ release from the sarcoplasmic reticulum (SR). It is currently disputed whether Ca 2+ release from RyRs could sustain viable pacemaker activity provided preserved SR Ca 2+ content. While working myocytes express L-type Ca v 1.2 channels to maintain SR Ca 2+ content, SAN cells express also L-type Ca v 1.3 and T-type Ca v 3.1 channels to generate pacemaking. We used mutant mice carrying concomitant ablation of Ca v 1.3 and Ca v 3.1 (Ca v 1.3 −/− /Ca v 3.1 −/− ) to study the importance of these channels in automaticity. We also investigated the role of f-HCN channels and RyR-dependent Ca 2+ release in residual pacemaker activity of mutant mice. We employed in vivo telemetric recordings of heart rate (HR) in Ca v 1.3 −/− , Ca v 3.1 −/− and Ca v 1.3 −/− /Ca v 3.1 −/− mice. We studied the consequences of pharmacologic inhibition of f-HCN and TTX-sensitive Na + channels in mutant mice using Langendorff perfused hearts or optical mapping (OM) of the pacemaker impulse in intact SAN preparations (SANs). Ca v ablation reduced HR in mice: Ca v 3.1 −/− (−7.6%, n = 11), Ca v 1.3 −/− (−24.4%, n = 8), Ca v 1.3 −/− /Ca v 3.1 −/− (−35%, n = 11). In OM experiments on SANs, concomitant inhibition of f-HCN and Na v 1.1 channels slowed pacemaking in wild-type (−48%, n = 7) and Ca v 3.1 −/− (−37%, n = 7), while arresting automaticity in 4/6 of Ca v 1.3 −/− , 3/6 of Ca v 1.3 −/− /Ca v 3.1 −/− . When present, residual pacemaking was reduced by 82%. Similar results were obtained using isolated Ca v 1.3 −/− /Ca v 3.1 −/− pacemaker cells were automaticity arrested in 5/9 cells tested, or was reduced by 80% in 4/9 cells. Heart automaticity is primarily generated by Ca v 1.3 and f-HCN channels. RyR-dependent Ca 2+ release cannot sustain automaticity following concomitant targeting of Ca v 1.3 and f-HCN channels.

RyR2R420Q catecholaminergic polymorphic ventricular tachycardia mutation induces bradycardia by disturbing the coupled clock pacemaker mechanism

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

A Sexy Approach to Pacemaking

Introduction: The sinoatrial node (SAN) is the primary pacemaker of the heart. Clinical evidence shows that SAN function differs between sexes. Males have slower resting (extrinsic and intrinsic) heart rates, and longer corrected SAN recovery time compared with females. Hypothesis: Sex based differences may be due to SAN ion channel differences. Therefore, we investigated functional behaviour and molecular makeup of SANs in male and female rats. Methods: Intracellular action potential recordings were obtained using sharp microelectrode technique (n=5 each). Gene expression was measured with quantitative PCR (n=6 each). Results: The amplitude of the action potential was significantly higher in male SANs (45.3 ± 2.4) compared with female (31.7 ± 2.5). The upstroke velocity (dV/dtmax) was significantly faster in female SANs (16.4 ± 1.6) compared with male (9.1 ± 1.5). Out of 96 transcripts investigated, two were significantly different between the sexes. L-Type Ca2+ channel, Cav1.3, and acetylcholine activated K+ channel, Kir3.1, mRNA levels were significantly higher in female SANs (66.6% and 45.6% respectively) compared with male. Immunolabelling for Cav1.3 showed levels were significantly higher in female SANs compared with male (18.6%, n=6 each). Inhibition of Cav1.3 with nifedipine (5μM) showed greater effect on the beating rate of male SANs compared with female (43%). A non-coding RNA, miR-139-3p (predicted by Ingenuity IPA software to affect Cav1.3) showed greater effect on the beating rate of male SAN preparations compared with female. Mathematical modelling showed that a higher density of Cav1.3 (as in the case of females) has a protective role from sinoatrial node arrhythmias. Conclusion: This study identified functional and molecular differences in key pacemaker ion channels in SAN between sexes. These differences advance our understanding of gender differences in cardiac electrophysiology and may have implications for gender specific design of anti-arrhythmic drugs and biological pacemakers.

Gap junction modifiers regulate electrical activities of the sinoatrial node and pulmonary vein: Therapeutic implications in atrial arrhythmogenesis

BackgroundGap junction (GJ) dysfunctions predispose cardiac tissues to various arrhythmias. Sinoatrial node (SAN) and pulmonary veins (PVs) are closely related atrial dysrhythmia. This study evaluated whether GJ modifications modulate SAN and PVs electrical activities. MethodsConventional microelectrodes were used to record action potentials in isolated rabbit SAN, PVs, and connected PV-SAN tissue preparations before and after heptanol (GJ inhibitor) and PQ1 (GJ enhancer) administration with and without isoproterenol. A whole-cell patch clamp was used to record the electrical activities before and after heptanol in single SAN and PV cardiomyocytes. ResultsHeptanol (1, 3, and 10μM) reduced the spontaneous beating rates of isolated SAN preparations but not PVs. Heptanol (10μM) decelerated the SAN leading rhythm in the PV-SAN preparations and induced PV burst firings without (3 of 6, 50%) and with (6 of 6, 100%) isoproterenol (1μM). Heptanol (10μM) also reduced the spontaneous beating rates in single SAN cardiomyocyte, but not PV cardiomyocyte, with a decreased pacemaker current. PQ1 (50 and 500nM) treatment did not change the spontaneous beating rates in isolated SAN and PV preparations. In the connected PV-SAN preparations, PQ1 (500nM) did not induce any PV firing even having additional isoproterenol treatment (1μM). Moreover, PQ1 (500nM) prevented heptanol-induced electrical changes in SAN and PVs preparations. ConclusionGJ dysfunction modulates SAN and PV electrical activity, which may contribute to atrial arrhythmogenesis. GJ enhancer has a therapeutic potential in SAN dysfunction and atrial arrhythmogenesis.

L-type Cav1.3 channels regulate ryanodine receptor-dependent Ca2+ release during sino-atrial node pacemaker activity.

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

Selective and non-selective non-steroidal anti-inflammatory drugs differentially regulate pulmonary vein and atrial arrhythmogenesis

BackgroundNon-steroidal anti-inflammatory drugs (NSAIDs) increase the risk of atrial fibrillation (AF). This study investigated whether selective and non-selective NSAIDs differentially regulate the arrhythmogenesis of pulmonary veins and atria. MethodsConventional microelectrodes were used to record action potentials (APs) in isolated rabbit PVs, sinoatrial node (SAN), left atrium (LA), and right atrium (RA) preparations before and after celecoxib or indomethacin administration. A whole-cell patch clamp was used to record the sodium–calcium exchanger (NCX) current, L-type calcium current (ICa-L), and late sodium current (INa-late) before and after celecoxib administration in isolated PV cardiomyocytes. ResultsCelecoxib (0.3, 1, and 3μM) reduced PV spontaneous beating rates, and induced delayed afterdepolarizations and burst firings in four of eight PV preparations (50%, p<0.05). Celecoxib also reduced SAN beating rates and decreased AP durations (APDs) in RA and LA, but did not change the resting membrane potential. Indomethacin (0.3, 1, 3, and 10μM) changed neither the PV or SAN beating rates nor RA APDs, but it reduced LA APDs. Celecoxib (3μM) significantly increased the NCX current and decreased the ICa-L, but did not change the INa-late. Ranolazine (10μM) suppressed celecoxib (3μM)-induced PV burst firings in 6 (86%, p<0.05) of 7 PVs. KB-R7943 (10μM) suppressed celecoxib (3μM)-induced PV burst firings in 5 (71%, p<0.05) of 7 PVs. ConclusionsSelective and non-selective NSAIDs differentially modulate PV and atrial electrophysiological characteristics. Celecoxib increased PV triggered activity through enhancement of the NCX current, which contributed to its arrhythmogenesis.