Proarrhythmic Activity Research Articles

Circadian rhythm in pro-arrhythmic activity of pulmonary vein cardiomyocytes

Abstract Introduction Cardiomyocytes in the pulmonary vein (PV) sleeves are a major source of ectopic activity driving atrial fibrillation (AF) and episodes of AF in patients are more prevalent at night. While it is known that circadian clocks within the heart regionally control 24-hour variation in pacemaking and ventricular repolarisation, the mechanisms underlying the nighttime preponderance in AF are unknown. Purpose This study addresses the hypothesis that circadian rhythms exist in the pro-arrhythmic activity of PV cardiomyocytes. Methods Male Wistar rats maintained in a 24-hour cycle of 12-hr light/dark (lights-on at Zeitgeber time, ZT=0; lights-off, ZT12) were subject to terminal general anaesthesia (140 mg/kg Na pentobarbital i.p.) at ZT=0, 6, 12 or 18 hr and the hearts removed. RNA was extracted from left atrial (LA) appendage (LAA) and proximal PV at the LA/PV junction (3 rats per ZT) and RNA sequencing conducted. Reads were mapped to the rat genome, counts normalised and models in which ZT was or was not included as a factor compared (Likelihood Ratio Test, adjusted-P<0.01). Whole-cell current clamp recordings were made from cardiomyocytes isolated from the proximal PV (n=308) and the LAA (n=264) (N=74 rats). The effects of noradrenaline (NA, 1 µM) and acetylcholine (ACh, 1 µM) were examined. Data were plotted against the ZT of the time of recording and fitted to a sine wave to establish circadian rhythmicity (P<0.05, extra-sum-of-squares F-test). The effect of ≥24 hr constant dark in the period immediately before experiment was examined. Data are reported as mean ± standard error. Results The expression of 1368 genes varied significantly with ZT, including circadian clock components (e.g. Bmal1, Per1). PV cells were larger than LAA cells (73±1.6 pF vs 53±1.3 pF, P<0.0001) and had more depolarised resting membrane potential (-69±0.2 mV vs -72±0.1 mV, P<0.0001). Both cell types showed circadian variation in action potential duration at 90% repolarisation (APD90) and frequency of pro-arrhythmic activity. Pro-arrhythmic activity was greatest in PV cells and the frequency was greater during the rest phase (ZT0-12) in both cell types. In contrast, the circadian rhythm in APD90 differed between cell type, with the longest APD­­90 recorded during the active phase in PV cells (ZT12-24) but during the rest phase in LAA cells. Pro-arrhythmic activity was increased by NA and decreased by ACh in both cell types with maximal effect during the rest phase. Conclusion Circadian variation in APD90 and proarrhythmic-activity has been demonstrated in isolated proximal PV and LAA cardiomyocytes, with differences in rhythm between the two cell types. RNA sequencing suggests the presence of peripheral clocks in LAA and PV cardiomyocytes. Understanding these mechanisms is important in development of future therapeutic options for AF.

Advanced In Vitro Screening of New Drugs for Proarrhythmic Activity

Advanced In Vitro Screening of New Drugs for Proarrhythmic Activity

Suprastin (Chloropyramine) Causes Proarrhythmic Deterioration of Excitation Conduction, Depolarization and Potentiates Adrenergic Automaticity in the Pulmonary Veins Myocardium.

A number of pharmacological drugs have side effects that contribute to the occurrence of atrial fibrillation, the most common type of cardiac rhythm disorders. The clinical use of antihistamines is widespread; however, information regarding their anti- and/or proarrhythmic effects is contradictory. In this work, we studied the effects and mechanisms of the potential proarrhythmic action of the first-generation antihistamine chloropyramine (Suprastin) in the atrial myocardium and pulmonary vein (PV) myocardial tissue. In PV, chloropyramine caused depolarization of the resting potential and led to reduction of excitation wave conduction. These effects are likely due to suppression of the inward rectifier potassium current (IK1). In presence of epinephrine, chloropyramine induced spontaneous automaticity in the PV and could not be suppressed by atrial pacing. Chloropyramine change functional characteristics of PV and contribute to occurrence of atrial fibrillation. It should be noted that chloropyramine does not provoke atrial tachyarrhythmias, but create conditions for their occurrence during physical exercise and sympathetic stimulation.

CaMKIIδ-dependent dysregulation of atrial Na+ homeostasis promotes pro-arrhythmic activity in an obstructive sleep apnea mouse model.

Obstructive sleep apnea (OSA) has been linked to various pathologies, including arrhythmias such as atrial fibrillation. Specific treatment options for OSA are mainly limited to symptomatic approaches. We previously showed that increased production of reactive oxygen species (ROS) stimulates late sodium current through the voltage-dependent Na+ channels via Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ), thereby increasing the propensity for arrhythmias. However, the impact on atrial intracellular Na+ homeostasis has never been demonstrated. Moreover, the patients often exhibit a broad range of comorbidities, making it difficult to ascertain the effects of OSA alone. We analyzed the effects of OSA on ROS production, cytosolic Na+ level, and rate of spontaneous arrhythmia in atrial cardiomyocytes isolated from an OSA mouse model free from comorbidities. OSA was induced in C57BL/6 wild-type and CaMKIIδ-knockout mice by polytetrafluorethylene (PTFE) injection into the tongue. After 8 weeks, their atrial cardiomyocytes were analyzed for cytosolic and mitochondrial ROS production via laser-scanning confocal microscopy. Quantifications of the cytosolic Na+ concentration and arrhythmia were performed by epifluorescence microscopy. PTFE treatment resulted in increased cytosolic and mitochondrial ROS production. Importantly, the cytosolic Na+ concentration was dramatically increased at various stimulation frequencies in the PTFE-treated mice, while the CaMKIIδ-knockout mice were protected. Accordingly, the rate of spontaneous Ca2+ release events increased in the wild-type PTFE mice while being impeded in the CaMKIIδ-knockout mice. Atrial Na+ concentration and propensity for spontaneous Ca2+ release events were higher in an OSA mouse model in a CaMKIIδ-dependent manner, which could have therapeutic implications.

Cardiovascular safety of non-steroidal anti-inflammatory drug use for gout: A Danish nationwide case-crossover study

Abstract Background Gout is associated with increased cardiovascular risk through inflammation and shared risk factors. Gout attacks are treated with a uric-lowering drug, such as allopurinol, and an anti-inflammatory drug, such as a non-steroidal anti-inflammatory drug (NSAID). Thus, NSAIDs could be both cardiovascular beneficial, due to their anti-inflammatory actions, and cardiovascular hazardous, due to their prothrombotic, hypertensive, and proarrhythmic actions. Yet, no study has examined the cardiovascular safety of NSAIDs for the treatment of gout. Purpose To examine the risk of major adverse cardiovascular events (MACEs) associated with NSAID use in gout patients. Methods We conducted a nationwide, population-based case-crossover study of all Danes ≥18 years of age with first-time gout during 1997–2020, who experienced a MACE (myocardial infarction, ischemic stroke, congestive heart failure, atrial fibrillation or flutter, or all-cause death) (n=103,522). The patients were identified either via a hospital diagnosis for gout (12%) or via a filling of an allopurinol prescription (88%). The exposures were use of NSAIDs overall and according to subtype (ibuprofen, naproxen, or diclofenac). An individual was considered exposed from the time of a filled prescription until the end of a use period, estimated from average use of two tablets per day. In the case-crossover design, we used four reference dates at 120, 180, 240, and 300 days before the outcome. An individual was considered exposed if the use period covered a reference date or the outcome date. We used the Mantel-Haenszel method to calculate odds ratios (ORs) with 95% confidence intervals (CIs) of the association between NSAID use and MACE. Results NSAID use was overall associated with a 15% decreased risk of MACE (OR=0.85, 95% CI: 0.83–0.87). The ORs of the association between NSAID use and the individual cardiovascular events were 0.91 (95% CI: 0.84–0.99) for myocardial infarction, 0.88 (95% CI: 0.81–0.95) for ischemic stroke, 0.89 (95% CI: 0.83–0.94) for congestive heart failure, 0.80 (95% CI: 0.75–0.85) for atrial fibrillation or flutter, and 0.77 (95% CI: 0.75–0.80) for all-cause death. The decreased risk of MACE was observed for use of ibuprofen (OR=0.91, 95% CI: 0.88–0.95) and naproxen (OR=0.78, CI: 0.70–0.86), but not for use of diclofenac (OR=1.03, 95% CI: 0.97–1.09). Conclusions In patients with first-time gout, use of ibuprofen and naproxen was associated with a slightly decreased cardiovascular risk, whereas diclofenac was not.

Pro-arrhythmic effect of escitalopram and citalopram at serum concentrations commonly observed in older patients – a study based on a cohort of 19,742 patients

For a decade, patients have been advised against using high citalopram- and escitalopram-doses due to risk for ventricular arrhythmia and cardiac arrest. Still, these drugs are widely used to treat depression and anxiety especially in older patients. It is unclear why they are cardiotoxic and at what serum concentrations patients are at risk for arrhythmias. Thus, how many patients that are at risk for iatrogenic cardiac arrest is unknown. We studied the arrhythmogenic effects of citalopram, escitalopram and their metabolites on human cardiomyocytes. Concentrations showing pro-arrhythmic activity were compared with observed drug and metabolite serum concentrations in a cohort of 19,742 patients (age 12-105 years) using escitalopram or citalopram in Norway (2010-2019). As arrhythmia-risk is related to maximum serum concentration, this was simulated for different age-groups from the escitalopram patient material. Therapeutic concentrations of both citalopram and escitalopram but not their metabolites showed pro-arrhythmic changes in the human cardiac action potential. Due to age-dependent reduction of drug clearance, the proportion of patients above threshold for arrhythmia-risk increased with age. 20% of patients >65 years were predicted to reach potentially pro-arrhythmic concentrations, following intake of 10mg escitalopram. All patients that are using escitalopram or citalopram and have genetic disposition for acquired long-QT syndrome, are >65 years, are using additional pro-arrhythmic drugs or have predisposition for arrhythmias, should be monitored with therapeutic drug monitoring (TDM) to avoid exposure to potentially cardiotoxic concentrations. Serum concentrations should be kept below 100nM, to reduce arrhythmia-risk. This study was funded by The Research Council of Norway (project number: 324062).

Lysosomal calcium loading promotes spontaneous calcium release by potentiating ryanodine receptors

Spontaneous calcium release by ryanodine receptors (RyRs) due to intracellular calcium overload results in delayed afterdepolarizations, closely associated with life-threatening arrhythmias. In this regard, inhibiting lysosomal calcium release by two-pore channel 2 (TPC2) knockout has been shown to reduce the incidence of ventricular arrhythmias under β-adrenergic stimulation. However, mechanistic investigations into the role of lysosomal function on RyR spontaneous release remain missing. We investigate the calcium handling mechanisms by which lysosome function modulates RyR spontaneous release, and determine how lysosomes are able to mediate arrhythmias by its influence on calcium loading. Mechanistic studies were conducted using a population of biophysically detailed mouse ventricular models including for the first time modeling of lysosomal function, and calibrated by experimental calcium transients modulated by TPC2. We demonstrate that lysosomal calcium uptake and release can synergistically provide a pathway for fast calcium transport, by which lysosomal calcium release primarily modulates sarcoplasmic reticulum calcium reuptake and RyR release. Enhancement of this lysosomal transport pathway promoted RyR spontaneous release by elevating RyR open probability. In contrast, blocking either lysosomal calcium uptake or release revealed an antiarrhythmic impact. Under conditions of calcium overload, our results indicate that these responses are strongly modulated by intercellular variability in L-type calcium current, RyR release, and sarcoplasmic reticulum calcium-ATPase reuptake. Altogether, our investigations identify that lysosomal calcium handling directly influences RyR spontaneous release by regulating RyR open probability, suggesting antiarrhythmic strategies and identifying key modulators of lysosomal proarrhythmic action.

Input of expressed hERG current into a complex neuron in silico cardiac atrial cell: Implications for cardiotoxicity screening.

NEURON is the leading in silico software for generating computational models of excitable cells (www.neuron.yale/edu/neuron/). We have combined our real-time, dynamic clamp system with offline NEURON models allowing for interfacing of living cells with NEURON models during electrophysiological recording. For these studies, we employed a complex in silico atrial cardiomyocyte (Courtemanche et al., 1998; Jacobson, 1998). In Proof of Concept studies, the cardiac atrial cardiomyocyte IKr model was replaced with the FDA ORD HERG model (Li et al., 2017) via dynamic clamp. The in silico atrial cardiomyocyte AP displayed a spike and dome morphology with a resting membrane potential of −82±0.004 mV, an amplitude of 104±2 mV, a dV/dt of 183±8 V/s and an APD90 of 303±2 ms (n=8). The NEURON in silico cardiomyocyte IKr was replaced with the FDA ORD HERG model and current magnitude scaled such that the APD90 value was ∼300 ms. The AP also displayed a spike and dome morphology with a resting membrane potential of −82±0.005 mV, an amplitude of 99±1 mV, a dV/dt of 151±7 V/s and an APD90 of 305±2 ms (n=8). Virtual drug block with 10 nM dofetilide significantly prolonged the AP90 to 350±3 ms (p<0.001). The effect of blocking real HERG current expressed in HEK cells on in silico atrial cardiomyocyte AP morphology will be examined. Future studies will measure the effect of blocking real HERG current on AP morphology using the FDA CiPA's O’Hara-Rudy in silico ventricular cardiomyocyte. This assay will have utility in screening drugs for proarrhythmic activity where the measured output of HERG block is AP behavior. Importantly, this assay can readily be implemented on automated patch clamp instrumentation with our dynamic clamp system.

CaMKII-Dependent Contractile Dysfunction and Pro-Arrhythmic Activity in a Mouse Model of Obstructive Sleep Apnea.

Left ventricular contractile dysfunction and arrhythmias frequently occur in patients with sleep-disordered breathing (SDB). The CaMKII-dependent dysregulation of cellular Ca homeostasis has recently been described in SDB patients, but these studies only partly explain the mechanism and are limited by the patients' heterogeneity. Here, we analyzed contractile function and Ca homeostasis in a mouse model of obstructive sleep apnea (OSA) that is not limited by confounding comorbidities. OSA was induced by artificial tongue enlargement with polytetrafluorethylene (PTFE) injection into the tongue of wildtype mice and mice with a genetic ablation of the oxidative activation sites of CaMKII (MMVV knock-in). After eight weeks, cardiac function was assessed with echocardiography. Reactive oxygen species (ROS) and Ca transients were measured using confocal and epifluorescence microscopy, respectively. Wildtype PTFE mice exhibited an impaired ejection fraction, while MMVV PTFE mice were fully protected. As expected, isolated cardiomyocytes from PTFE mice showed increased ROS production. We further observed decreased levels of steady-state Ca transients, decreased levels of caffeine-induced Ca transients, and increased pro-arrhythmic activity (defined as deviations from the diastolic Ca baseline) only in wildtype but not in MMVV PTFE mice. In summary, in the absence of any comorbidities, OSA was associated with contractile dysfunction and pro-arrhythmic activity and the inhibition of the oxidative activation of CaMKII conveyed cardioprotection, which may have therapeutic implications.

Critical Link between Calcium Regional Heterogeneity and Atrial Fibrillation Susceptibility in Sheep Left Atria.

Atrial fibrillation is the most sustained form of arrhythmia in the human population that leads to important electrophysiological and structural cardiac remodeling as it progresses into a chronic form. Calcium is an established key player of cellular electrophysiology in the heart, yet to date, there is no information that maps calcium signaling across the left atrium. The aim of this study is to determine whether calcium signaling is homogenous throughout the different regions of the left atrium. This work tests the hypothesis that differences across the healthy left atrium contribute to a unique, region-dependent calcium cycling and participates in the pro-arrhythmic activity during atrial fibrillation. An animal model relevant to human cardiac function (the sheep) was used to characterize both the electrical activity and the calcium signaling of three distinct left atrium regions (appendage, free wall and pulmonary veins) in control conditions and after acetylcholine perfusion (5 μM) to induce acute atrial fibrillation. High-resolution dual calcium-voltage optical mapping on the left atria of sheep was performed to explore the spatiotemporal dynamics of calcium signaling in relation to electrophysiological properties. Action potential duration (at 80% repolarization) was not significantly different in the three regions of interest for the three pacing sites. In contrast, the time to 50% calcium transient decay was significantly different depending on the region paced and recorded. Acetylcholine perfusion and burst pacing-induced atrial fibrillation when pulmonary veins and appendage regions were paced but not when the free wall region was. Dantrolene (a ryanodine receptor blocker) did not reduce atrial fibrillation susceptibility. These data provide the first evidence of heterogenous calcium signaling across the healthy left atrium. Such basal regional differences may be exacerbated during the progression of atrial fibrillation and thus play a crucial role in focal arrhythmia initiation without ryanodine receptor gating modification.

Modeling incomplete penetrance in long QT syndrome type 3 through ion channel heterogeneity: an in silico population study.

Many cardiac diseases are characterized by an increased late sodium current, including heart failure, hypertrophic cardiomyopathy, and inherited long QT syndrome type 3 (LQT3). The late sodium current in LQT3 is caused by a gain-of-function mutation in the voltage-gated sodium channel Nav1.5. Despite a well-defined genetic cause of LQT3, treatment remains inconsistent because of incomplete penetrance of the mutation and variability of antiarrhythmic efficacy. Here, we investigate the relationship between LQT3-associated mutation incomplete penetrance and variability in ion channel expression, simulating a population of 1,000 individuals with the O'Hara-Rudy model of the human ventricular myocyte. We first simulate healthy electrical activity (i.e., in the absence of a mutation) and then incorporate heterozygous expression for three LQT3-associated mutations (Y1795C, I1768V, and ΔKPQ), to directly compare the effects of each mutation on individuals across a diverse population. For all mutations, we find that susceptibility, defined by either the presence of an early afterdepolarization (EAD) or prolonged action potential duration (APD), primarily depends on the balance between the conductance of IKr and INa, for which individuals with a higher IKr-to-INa ratio are less susceptible. Furthermore, we find distinct differences across the population, observing individuals susceptible to zero, one, two, or all three mutations. Individuals tend to be less susceptible with an appropriate balance of repolarizing currents, typically via increased IKs or IK1. Interestingly, the more critical repolarizing current is mutation specific. We conclude that the balance between key currents plays a significant role in mutant-specific presentation of the disease phenotype in LQT3.NEW & NOTEWORTHY An in silico population approach investigates the relationship between variability in ion channel expression and gain-of-function mutations in the voltage-gated sodium channel associated with the congenital disorder long QT syndrome type 3 (LQT3). We find that ion channel variability can contribute to incomplete penetrance of the mutation, with mutant-specific differences in ion channel conductances leading to susceptibility to proarrhythmic action potential duration prolongation or early afterdepolarizations.

InsP3R-RyR Ca2+ channel crosstalk facilitates arrhythmias in the failing human ventricle.

Dysregulated intracellular Ca2+ handling involving altered Ca2+ release from intracellular stores via RyR channels underlies both arrhythmias and reduced function in heart failure (HF). Mechanisms linking RyR dysregulation and disease are not fully established. Studies in animals support a role for InsP3 receptor Ca2+ channels (InsP3R) in pathological alterations in cardiomyocyte Ca2+ handling but whether these findings translate to the divergent physiology of human cardiomyocytes during heart failure is not determined. Using electrophysiological and Ca2+ recordings in human ventricular cardiomyocytes, we uncovered that Ca2+ release via InsP3Rs facilitated Ca2+ release from RyR and induced arrhythmogenic delayed after depolarisations and action potentials. InsP3R-RyR crosstalk was particularly increased in HF at RyR clusters isolated from the T-tubular network. Reduced SERCA activity in HF further facilitated the action of InsP3. Nanoscale imaging revealed co-localisation of InsP3Rs with RyRs in the dyad, which was increased in HF, providing a mechanism for augmented Ca2+ channel crosstalk. Notably, arrhythmogenic activity dependent on InsP3Rs was increased in tissue wedges from failing hearts perfused with AngII to promote InsP3 generation. These data indicate a central role for InsP3R-RyR Ca2+ signalling crosstalk in the pro-arrhythmic action of GPCR agonists elevated in HF and the potential for their therapeutic targeting.

Oxidative activation of CaMKII mediates arrhythmias and contractile dysfunction in an obstructive sleep apnea mouse model

Abstract Background/Introduction Atrial arrhythmias frequently occur in patients with obstructive sleep apnea (OSA), but the underlying mechanisms remain insufficiently understood. We recently demonstrated that CaMKII-dependent pro-arrhythmic activity is increased in patients with OSA, but the mechanisms of CaMKII activation remain unknown. Interestingly, OSA can lead to increased reactive oxygen species production, which may facilitate CaMKII activation by oxidation at methionine residues 281/282. Purpose We tested if oxidation of CaMKII is involved in the development of arrhythmias and contractile dysfunction in vitro and in vivo in a mouse model of obstructive sleep apnea by tongue enlargement. Methods Experiments were performed using 41 wild-type (WT) and 25 genetically modified mice lacking oxidative CaMKII activation due to mutation of methionine 281/282 to valine (MMVV mice). Polytetrafluorethylene (PTFE, 100 μl) was injected into the tongue of 23 WT and 12 MMVV mice to induce OSA by sustained tongue enlargement as previously established. Whole body plethysmography was performed to confirm success of intervention. Echocardiography was performed at baseline and after 8 weeks to assess left-ventricular function. After 8 weeks, isolated atrial and ventricular cardiomyocytes were incubated with the Ca-sensitive dye FURA-2 AM (5 μM, 15 min) and analyzed with epifluorescence microscopy under regular electrical field stimulation (1 Hz). Results PTFE injection resulted in an increased frequency of inspiratory flow limitations (IFLs/h) from 27.4±5.95 to 59.6±6.22 in PTFE mice (p&amp;lt;0.001, fig. 1A). Interestingly, in WT PTFE mice, left ventricular ejection fraction (LVEF, in %) was reduced at 8 weeks post procedure from 57.0±1.36 to 51.5±1.85 (p=0.001, fig. 1B). Importantly, mice lacking oxidative CaMKII activation were protected from such decline in contractile function, and LVEF was higher at 8 weeks vs. WT PTFE (p&amp;lt;0.001, fig. 1B). Congruent with deterioration of contractility in vivo, the Ca transient amplitude after 30s pause (normalized to steady-state before pause) was decreased in ventricular cardiomyocytes of WT PTFE mice indicating increased SR Ca leak (p for interaction genotype x PTFE = 0.014, fig. 1C). In contrast to WT, the post-pause ratio was increased in MMVV PTFE mice (p=0.018, fig. 1C). In addition to contractile function, cellular arrhythmic events were analyzed as non-stimulated pro-arrhythmic events (NSEs) during steady-state electrical field stimulation at 1 Hz (fig. 1D, red arrows). NSE frequency was increased in atrial cardiomyocytes from WT PTFE mice with 0.050±0.005 compared to 0.018±0.003 s–1 in WT CTRL (p&amp;lt;0.001, fig. 1D). Intriguingly, MMVV mice were protected from NSEs after PTFE treatment (0.020±0.004, p&amp;lt;0.001 vs. WT PTFE, Fig. 1D). Conclusion In a mouse model of obstructive sleep apnea, contractile dysfunction and pro-arrhythmic activity are modulated by oxidative CaMKII activation, which may have therapeutic implications. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft; Medical Faculty at University of Regensburg

SAR296968, a Novel Selective Na+/Ca2+ Exchanger Inhibitor, Improves Ca2+ Handling and Contractile Function in Human Atrial Cardiomyocytes.

Background: In reverse-mode, cardiac sodium-calcium exchanger (NCX) can increase the cytoplasmic Ca2+ concentration in response to high intracellular Na+ levels, which may contribute to diastolic contractile dysfunction. Furthermore, increased spontaneous Ca2+ release from intracellular stores can activate forward mode NCX. The resulting transient inward current causes delayed afterdepolarization (DAD)-dependent arrhythmias. Moreover, recently, NCX has been associated with impaired relaxation and reduced cardiac function in heart failure with preserved ejection fraction (HFpEF). Since NCX is upregulated in human chronic atrial fibrillation (AF) as well as heart failure (HF), specific inhibition may have therapeutic potential. Objective: We tested the antiarrhythmic, lusitropic and inotropic effects of a novel selective NCX-inhibitor (SAR296968) in human atrial myocardium. Methods and Results: Right atrial appendage biopsies of 46 patients undergoing elective cardiac surgery in a predominant HFpEF cohort (n = 24/46) were investigated. In isolated human atrial cardiomyocytes, SAR296968 reduced the frequency of spontaneous SR Ca2+ release events and increased caffeine transient amplitude. In accordance, in isolated atrial trabeculae, SAR296968 enhanced the developed tension after a 30 s pause of electrical stimulation consistent with reduced diastolic sarcoplasmic reticulum (SR) Ca2+ leak. Moreover, compared to vehicle, SAR296968 decreased steady-state diastolic tension (at 1 Hz) without impairing developed systolic tension. Importantly, SAR296968 did not affect the safety parameters, such as resting membrane potential or action potential duration as measured by patch clamp. Conclusion: The novel selective NCX-inhibitor SAR296968 inhibits atrial pro-arrhythmic activity and improves diastolic and contractile function in human atrial myocardium, which may have therapeutic implications, especially for treatment of HFpEF.

Aminophylline Induces Two Types of Arrhythmic Events in Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Cardiac side effects of some pulmonary drugs are observed in clinical practice. Aminophylline, a methylxanthine bronchodilator with documented proarrhythmic action, may serve as an example. Data on the action of aminophylline on cardiac cell electrophysiology and contractility are not available. Hence, this study was focused on the analysis of changes in the beat rate and contraction force of human pluripotent stem cell–derived cardiomyocytes (hPSC-CMs) and HL-1 cardiomyocytes in the presence of increasing concentrations of aminophylline (10 µM–10 mM in hPSC-CM and 8–512 µM in HL-1 cardiomyocytes). Basic biomedical parameters, namely, the beat rate (BR) and contraction force, were assessed in hPSC-CMs using an atomic force microscope (AFM). The beat rate changes under aminophylline were also examined on the HL-1 cardiac muscle cell line via a multielectrode array (MEA). Additionally, calcium imaging was used to evaluate the effect of aminophylline on intracellular Ca2+ dynamics in HL-1 cardiomyocytes. The BR was significantly increased after the application of aminophylline both in hPSC-CMs (with 10 mM aminophylline) and in HL-1 cardiomyocytes (with 256 and 512 µM aminophylline) in comparison with controls. A significant increase in the contraction force was also observed in hPSC-CMs with 10 µM aminophylline (a similar trend was visible at higher concentrations as well). We demonstrated that all aminophylline concentrations significantly increased the frequency of rhythm irregularities (extreme interbeat intervals) both in hPSC-CMs and HL-1 cells. The occurrence of the calcium sparks in HL-1 cardiomyocytes was significantly increased with the presence of 512 µM aminophylline. We conclude that the observed aberrant cardiomyocyte response to aminophylline suggests an arrhythmogenic potential of the drug. The acquired data represent a missing link between the arrhythmic events related to the aminophylline/theophylline treatment in clinical practice and describe cellular mechanisms of methylxanthine arrhythmogenesis. An AFM combined with hPSC-CMs may serve as a robust platform for direct drug effect screening.

Antiviral and anti-inflammatory drugs to combat COVID-19: Effects on cardiac ion channels and risk of ventricular arrhythmias.

Introduction: Drugs with no indication for the treatment of cardiovascular diseases (e.g., drugs employed to treat COVID-19) can increase the risk of arrhythmias. Of interest, a six-fold increase in the number of arrhythmic events was reported in patients with severe COVID-19. In this study, we reviewed (i) the pro-arrhythmic action of drugs given to patients with COVID-19 infection, and (ii) the effects of inflammatory cytokines on cardiac ion channels and possible generation of arrhythmias. Methods: We conducted a literature search on the drugs with purported or demonstrated efficacy against COVID-19 disease, emphasizing the mechanisms by which anti-COVID-19 drugs and inflammatory cytokines interfere with cardiac ion channels. Results: Antibiotics (azithromycin), antimalarials (hydroxychloroquine, chloroquine), antivirals (ritonavir/lopinavir, atazanavir), and some of the tyrosine kinase inhibitors (vandetanib) could induce long QT and increase risk for ventricular arrhythmias. The pro-arrhythmic action results from drug-induced inhibition of Kv11.1 (hERG) channels interfering with the repolarizing potassium IKr currents, leading to long QT and increased risk of triggered arrhythmias. At higher concentrations, these drugs may interfere with IKs, IK1, and/or Ito potassium currents, and even inhibit sodium (INa) and calcium (ICa) currents, inducing additional cardiac toxicity. Ibrutinib, an inhibitor of Bruton’s TK, increased the incidence of atrial fibrillation and ventricular tachycardia associated with a short QT interval. Inflammatory cytokines IL-6 and TNF-α inhibit IKr and Ito repolarizing potassium currents. High levels of inflammatory cytokines could contribute to the arrhythmic events. For remdesivir, favipiravir, dexamethasone, tocilizumab, anakinra, baricitinib, and monoclonal antibodies (bamlanivimab, etesevimab, and casirivimab), no evidence supports significant effects on cardiac ion channels, changes in the QT interval, and increased risk for ventricular arrhythmias. Conclusion: This study supports the concept of hERG channel promiscuity. Different drug classes given to COVID-19 patients might delay repolarization, and increase the risk of ventricular arrhythmias. The presence of comorbid pro-arrhythmic disease states, and elevated levels of pro-arrhythmic cytokines, could increase the risk of ventricular arrhythmias. Discontinuation of nonessential drugs and correction of electrolyte abnormalities could prevent severe ventricular arrhythmias. Altogether, the most effective therapies against COVID-19 (remdesivir, dexamethasone, monoclonal antibodies) lack pro-arrhythmic activity.

Central Sleep Apnea Is Associated with an Abnormal P-Wave Terminal Force in Lead V1 in Patients with Acute Myocardial Infarction Independent from Ventricular Function.

Sleep-disordered breathing (SDB) is highly prevalent in patients with cardiovascular disease. We have recently shown that an elevation of the electrocardiographic (ECG) parameter P wave terminal force in lead V1 (PTFV1) is linked to atrial proarrhythmic activity by stimulation of reactive oxygen species (ROS)-dependent pathways. Since SDB leads to increased ROS generation, we aimed to investigate the relationship between SDB-related hypoxia and PTFV1 in patients with first-time acute myocardial infarction (AMI). We examined 56 patients with first-time AMI. PTFV1 was analyzed in 12-lead ECGs and defined as abnormal when ≥4000 µV*ms. Polysomnography (PSG) to assess SDB was performed within 3–5 days after AMI. SDB was defined by an apnea-hypopnea-index (AHI) >15/h. The multivariable regression analysis showed a significant association between SDB-related hypoxia and the magnitude of PTFV1 independent from other relevant clinical co-factors. Interestingly, this association was mainly driven by central but not obstructive apnea events. Additionally, abnormal PTFV1 was associated with SDB severity (as measured by AHI, B 21.495; CI [10.872 to 32.118]; p < 0.001), suggesting that ECG may help identify patients suitable for SDB screening. Hypoxia as a consequence of central sleep apnea may result in atrial electrical remodeling measured by abnormal PTFV1 in patients with first-time AMI independent of ventricular function. The PTFV1 may be used as a clinical marker for increased SDB risk in cardiovascular patients.

Increased NaV1.8 expression in patients with sleep-disordered breathing induces pro-arrhythmic activity

Abstract Background Sleep-disordered breathing (SDB) is often associated with atrial fibrillation, but detailed mechanisms remain elusive. Interestingly, late Na current (late INa) has been shown to be increased in patients with SDB, while expression of cardiac Na channel NaV1.5 and peak Na current were decreased. Indeed, recent data demonstrated that enhanced NaV1.8-dependent late INa may also induce pro-arrhythmic activity. Purpose We tested whether Na-V1.8 expression and subsequent NaV1.8-dependent pro-arrhythmic activity are increased in patients with SDB. Methods We prospectively analysed 29 right atrial appendage biopsies of patients undergoing elective coronary artery bypass grafting. SDB was assessed using polygraphy in the preoperative night and an apnoea-hypopnea index (AHI) ≥15/h defined SDB. Micro-dissected atrial trabeculae were electrically field stimulated (at 1 Hz, 5 V for 50 ms, at 37°C) to elicit regular contractions. Trabecular arrhythmias were induced using 100 nM isoproterenol at [Ca]o of 3.5 mmol/L and pro-arrhythmic activity was scored from 0 (no arrhythmias) to 5 (salve). Sarcoplasmic reticulum Ca leak was estimated by the contractility after paused stimulation (at 2 Hz, normalized to before pause). To correlate functional and expression data for each individual patient, NaV1.8 mRNA expression was quantified in each trabeculum using qPCR. Results NaV1.8 mRNA expression was increased in patients with SDB, leading to a significant positive correlation with the severity of SDB (i.e. AHI, p=0.02, r2=0.22, Fig. 1A). Multivariate regression analysis revealed that this association was independent from age, sex, atrial fibrillation, heart failure, diabetes mellitus, and renal function (p=0.03, r2=0.35). Accordingly, selective NaV1.8 blockade with PF-01247324 (PF, 1 μM, 30 min) significantly improved post-pause contractility of isolated trabeculae from 1.69±0.31 to 2.95±0.54 in patients with SDB (p=0.001), whereas no significant improvement was observed in patients without SDB. This resulted in significant positive correlations between the PF-dependent improvement of post-pause contractility and both AHI (p=0.047, r2=0.19) and NaV1.8 mRNA expression (p=0.03, r2=0.17). Most importantly, we also observed a significant increase in arrhythmia severity in patients with SDB of 2.21±0.52 (vs. 1.00±0.49, p=0.03) that could be significantly reduced by selective NaV1.8 inhibition with PF to 0.25±0.18 (p=0.0008, Fig. 1B). In accordance, there was a significant positive correlation between arrhythmia severity and AHI (p=0.01, r2=0.28) that was abolished in the presence of PF (interaction analysis: p=0.ehab724.33141, r2=0.46). Conclusion In patients with SDB, enhanced NaV1.8 expression contribute to atrial pro-arrhythmic activity independent from comorbidities. Selective NaV1.8 inhibition may have therapeutic implications for patients with SDB. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Part of the study was supported by grants from Philips Respironics (Murrysville, PA 15668) and the Medical Faculty at the University of Regensburg. Figure 1

Increased CaMKII-dependent pro-arrhythmic activity in a novel mouse model of obstructive sleep apnoea

Abstract Background Obstructive sleep apnoea (OSA) is frequently associated with atrial arrhythmias, but detailed mechanisms remain elusive. Most recently, we found an increased CaMKII-dependent pro-arrhythmic activity in patients with sleep apnoea. Since patients suffer from various confounding comorbidities, we have developed a novel mouse model of OSA by tongue enlargement. Purpose We tested if mice with OSA exhibit increased atrial CaMKII-dependent pro-arrhythmic activity. Methods Polytetrafluorethylene (PTFE) was injected into the tongue of 12 wild-type (WT) and 10 CaMKII knock-out (CKO) mice. 9 WT and 9 CKO mice were used as control without PTFE injection. Inspiratory flow limitations and apnoeas were monitored during murine sleep phases by whole-body plethysmography (Buxco). After eight weeks, isolated atrial cardiomyocytes were incubated with the Ca-sensitive dye FURA-2 AM for 15 min. Regular Ca transients were elicited by electrical field stimulation (1 Hz, 20 V for 4 ms) using epifluorescence microscopy. Pro-arrhythmic non-stimulated events were defined as deviations from diastolic Ca baseline between two stimulated Ca transients. Results Sonographic measurements revealed a significant increase in mean tongue diameter from (in mm) 3.7±0.1 to 5.1±0.1 after PTFE injection (n=23, p&amp;lt;0.0001). There was a significant correlation between magnitude of tongue diameter and frequency of apnoeas in OSA mice (p=0.046, r2=0.19, Fig. 1A). Interestingly, we observed a significantly increased frequency of pro-arrhythmic events of (in s–1) 0.06±0.01 in WT OSA mice compared to 0.02±0.01 in WT control mice (p=0.047, Fig. 1B). Similar results were observed at higher stimulation frequencies (2 and 4 Hz). There was a significant correlation of pro-arrhythmic events with inspiratory flow limitations (p=0.03, r2=0.24, Fig. 1C) and with the frequency of apnoeas by strong trend (p=0.06, r2=0.18). In contrast, no increase in atrial pro-arrhythmic events was observed in CKO mice after PTFE injection (for CKO mice after PTFE vs. CKO mice without PTFE, 0.03±0.01 s–1 vs. 0.03±0.01 s–1, p=0.89, Fig. 1B). Accordingly, the correlations between pro-arrhythmic events and both inspiratory flow limitations (p=0.36, r2=0.05, Fig. 1C) and apnoeas (p=0.82, r2=0.004) were completely abolished in CKO mice. Conclusion In a novel mouse model of obstructive sleep apnoea, atrial pro-arrhythmic activity was increased in a CaMKII-dependent fashion, which may have therapeutic implications. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Benedikt Schaner / Mr., this work is supported by a research grant of the German Cardiac Society (DGK); Stefan Wagner / Professor, was funded by DFG grants Figure 1

Cardiovascular Effects of Epinephrine During Experimental Hypothermia (32°C) With Spontaneous Circulation in an Intact Porcine Model

Aims: Rewarming from accidental hypothermia and therapeutic temperature management could be complicated by cardiac dysfunction. Although pharmacologic support is often applied when rewarming these patients, updated treatment recommendations are lacking. There is an underlying deficiency of clinical and experimental data to support such interventions and this prevents the development of clinical guidelines. Accordingly, we explored the clinical effects of epinephrine during hypothermic conditions.Materials and methods: Anesthetized pigs were immersion cooled to 32°C. Predetermined variables were compared at temperature/time-point baseline, after receiving 30 ng/kg/min and 90 ng/kg/min epinephrine infusions: (1) before and during hypothermia at 32°C, and after rewarming to 38°C (n = 7) and (2) a time-matched (5 h) normothermic control group (n = 5).Results: At 32°C, both stroke volume and cardiac output were elevated after 30 ng/kg/min administration, while systemic vascular resistance was reduced after 90 ng/kg/min. Epinephrine infusion did not alter blood flow in observed organs, except small intestine flow, and global O2 extraction rate was significantly reduced in response to 90 ng/kg/min infusion. Electrocardiographic measurements were unaffected by epinephrine infusion.Conclusion: Administration of both 30 ng/kg/min and 90 ng/kg/min at 32°C had a positive inotropic effect and reduced afterload. We found no evidence of increased pro-arrhythmic activity after epinephrine infusion in hypothermic pigs. Our experiment therefore suggests that β₁-receptor stimulation with epinephrine could be a favorable strategy for providing cardiovascular support in hypothermic patients, at core temperatures >32°C.