Jordi Heijman

Cellular and Molecular Electrophysiology of Atrial Fibrillation Initiation, Maintenance, and Progression

Atrial fibrillation (AF) is the most common clinically relevant arrhythmia and is associated with increased morbidity and mortality. The incidence of AF is expected to continue to rise with the aging of the population. AF is generally considered to be a progressive condition, occurring first in a paroxysmal form, then in persistent, and then long-standing persistent (chronic or permanent) forms. However, not all patients go through every phase, and the time spent in each can vary widely. Research over the past decades has identified a multitude of pathophysiological processes contributing to the initiation, maintenance, and progression of AF. However, many aspects of AF pathophysiology remain incompletely understood. In this review, we discuss the cellular and molecular electrophysiology of AF initiation, maintenance, and progression, predominantly based on recent data obtained in human tissue and animal models. The central role of Ca2+-handling abnormalities in both focal ectopic activity and AF substrate progression is discussed, along with the underlying molecular basis. We also deal with the ionic determinants that govern AF initiation and maintenance, as well as the structural remodeling that stabilizes AF-maintaining re-entrant mechanisms and finally makes the arrhythmia refractory to therapy. In addition, we highlight important gaps in our current understanding, particularly with respect to the translation of these concepts to the clinical setting. Ultimately, a comprehensive understanding of AF pathophysiology is expected to foster the development of improved pharmacological and nonpharmacological therapeutic approaches and to greatly improve clinical management.

Cellular and Molecular Mechanisms of Atrial Arrhythmogenesis in Patients With Paroxysmal Atrial Fibrillation

Background— Electrical, structural, and Ca2+-handling remodeling contribute to the perpetuation/progression of atrial fibrillation (AF). Recent evidence has suggested a role for spontaneous sarcoplasmic reticulum Ca2+-release events in long-standing persistent AF, but the occurrence and mechanisms of sarcoplasmic reticulum Ca2+-release events in paroxysmal AF (pAF) are unknown. Method and Results— Right-atrial appendages from control sinus rhythm patients or patients with pAF (last episode a median of 10–20 days preoperatively) were analyzed with simultaneous measurements of [Ca2+]i (fluo-3-acetoxymethyl ester) and membrane currents/action potentials (patch-clamp) in isolated atrial cardiomyocytes, and Western blot. Action potential duration, L-type Ca2+ current, and Na+/Ca2+-exchange current were unaltered in pAF, indicating the absence of AF-induced electrical remodeling. In contrast, there were increases in SR Ca2+ leak and incidence of delayed after-depolarizations in pAF. Ca2+-transient amplitude and sarcoplasmic reticulum Ca2+ load (caffeine-induced Ca2+-transient amplitude, integrated Na+/Ca2+-exchange current) were larger in pAF. Ca2+-transient decay was faster in pAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting increased SERCA2a function. In agreement, phosphorylation (inactivation) of the SERCA2a-inhibitor protein phospholamban was increased in pAF. Ryanodine receptor fractional phosphorylation was unaltered in pAF, whereas ryanodine receptor expression and single-channel open probability were increased. A novel computational model of the human atrial cardiomyocyte indicated that both ryanodine receptor dysregulation and enhanced SERCA2a activity promote increased sarcoplasmic reticulum Ca2+ leak and sarcoplasmic reticulum Ca2+-release events, causing delayed after-depolarizations/triggered activity in pAF. Conclusions— Increased diastolic sarcoplasmic reticulum Ca2+ leak and related delayed after-depolarizations/triggered activity promote cellular arrhythmogenesis in pAF patients. Biochemical, functional, and modeling studies point to a combination of increased sarcoplasmic reticulum Ca2+ load related to phospholamban hyperphosphorylation and ryanodine receptor dysregulation as underlying mechanisms.

Properties and ionic mechanisms of action potential adaptation, restitution, and accommodation in canine epicardium

Computational models of cardiac myocytes are important tools for understanding ionic mechanisms of arrhythmia. This work presents a new model of the canine epicardial myocyte that reproduces a wide range of experimentally observed rate-dependent behaviors in cardiac cell and tissue, including action potential (AP) duration (APD) adaptation, restitution, and accommodation. Model behavior depends on updated formulations for the 4-aminopyridine-sensitive transient outward current (I(to1)), the slow component of the delayed rectifier K(+) current (I(Ks)), the L-type Ca(2+) channel current (I(Ca,L)), and the Na(+)-K(+) pump current (I(NaK)) fit to data from canine ventricular myocytes. We found that I(to1) plays a limited role in potentiating peak I(Ca,L) and sarcoplasmic reticulum Ca(2+) release for propagated APs but modulates the time course of APD restitution. I(Ks) plays an important role in APD shortening at short diastolic intervals, despite a limited role in AP repolarization at longer cycle lengths. In addition, we found that I(Ca,L) plays a critical role in APD accommodation and rate dependence of APD restitution. Ca(2+) entry via I(Ca,L) at fast rate drives increased Na(+)-Ca(2+) exchanger Ca(2+) extrusion and Na(+) entry, which in turn increases Na(+) extrusion via outward I(NaK). APD accommodation results from this increased outward I(NaK). Our simulation results provide valuable insight into the mechanistic basis of rate-dependent phenomena important for determining the hearts response to rapid and irregular pacing rates (e.g., arrhythmia). Accurate simulation of rate-dependent phenomena and increased understanding of their mechanistic basis will lead to more realistic multicellular simulations of arrhythmia and identification of molecular therapeutic targets.

The value of basic research insights into atrial fibrillation mechanisms as a guide to therapeutic innovation: a critical analysis

Atrial fibrillation (AF) is an extremely common clinical problem associated with increased morbidity and mortality. Current antiarrhythmic options include pharmacological, ablation, and surgical therapies, and have significantly improved clinical outcomes. However, their efficacy remains suboptimal, and their use is limited by a variety of potentially serious adverse effects. There is a clear need for improved therapeutic options. Several decades of research have substantially expanded our understanding of the basic mechanisms of AF. Ectopic firing and re-entrant activity have been identified as the predominant mechanisms for arrhythmia initiation and maintenance. However, it has become clear that the clinical factors predisposing to AF and the cellular and molecular mechanisms involved are extremely complex. Moreover, all AF-promoting and maintaining mechanisms are dynamically regulated and subject to remodelling caused by both AF and cardiovascular disease. Accordingly, the initial presentation and clinical progression of AF patients are enormously heterogeneous. An understanding of arrhythmia mechanisms is widely assumed to be the basis of therapeutic innovation, but while this assumption seems self-evident, we are not aware of any papers that have critically examined the practical contributions of basic research into AF mechanisms to arrhythmia management. Here, we review recent insights into the basic mechanisms of AF, critically analyse the role of basic research insights in the development of presently used anti-AF therapeutic options and assess the potential value of contemporary experimental discoveries for future therapeutic innovation. Finally, we highlight some of the important challenges to the translation of basic science findings to clinical application.

Function and regulation of serine/threonine phosphatases in the healthy and diseased heart

Protein phosphorylation is a major control mechanism of a wide range of physiological processes and plays an important role in cardiac pathophysiology. Serine/threonine protein phosphatases control the dephosphorylation of a variety of cardiac proteins, thereby fine-tuning cardiac electrophysiology and function. Specificity of protein phosphatases type-1 and type-2A is achieved by multiprotein complexes that target the catalytic subunits to specific subcellular domains. Here, we describe the composition, regulation and target substrates of serine/threonine phosphatases in the heart. In addition, we provide an overview of pharmacological tools and genetic models to study the role of cardiac phosphatases. Finally, we review the role of protein phosphatases in the diseased heart, particularly in ventricular arrhythmias and atrial fibrillation and discuss their role as potential therapeutic targets.

IKs restricts excessive beat-to-beat variability of repolarization during beta-adrenergic receptor stimulation

In vivo studies have suggested that increased beat-to-beat variability of ventricular repolarization duration (BVR) is a better predictor of drug-induced torsades de pointes than repolarization prolongation alone. Cellular BVR and its dynamics before proarrhythmic events are poorly understood. We investigated differential responses of BVR in single myocytes during I(Ks) blockade versus I(Kr) blockade and late-I(Na) augmentation, under the influence of beta-adrenergic receptor stimulation. Transmembrane action potentials were recorded from isolated canine left-ventricular midmyocytes at various pacing rates. I(Ks) was blocked by HMR1556, I(Kr) by dofetilide. Late I(Na) was augmented by sea anemone toxin-II. Isoproterenol was added for beta-adrenergic receptor stimulation. BAPTA-AM buffered intracellular Ca(2+). SEA0400 partially inhibited the Na(+)-Ca(2+) exchanger. BVR was quantified as variability of action-potential duration at 90% repolarization: Sigma(|APD90; i+1 minus APD90; i|)/[nbeatsx radical2] for 30 consecutive action potentials. Baseline BVR was significantly increased by I(Kr) blockade and late-I(Na) augmentation, especially at slow pacing rates. beta-adrenergic stimulation restabilized these BVR changes. In contrast, I(Ks) blockade caused very little change in repolarization when compared to baseline conditions, but predisposed the myocyte to increased BVR during beta-adrenergic stimulation, especially at fast rates. BAPTA-AM and SEA0400 reduced this excessive BVR and eliminated early afterdepolarizations. In conclusion, beta-adrenergic receptor stimulation exaggerates BVR during I(Ks) blockade, indicating a BVR-stabilizing role of beta-adrenergic-sensitive I(Ks). Loss of I(Ks) plus overriding of Ca(2+)-dependent membrane currents, including inward Na(+)-Ca(2)(+) exchange current, conspire to proarrhythmic BVR under these conditions.

Determinants of Beat-to-Beat Variability of Repolarization Duration in the Canine Ventricular Myocyte: A Computational Analysis

Beat-to-beat variability of repolarization duration (BVR) is an intrinsic characteristic of cardiac function and a better marker of proarrhythmia than repolarization prolongation alone. The ionic mechanisms underlying baseline BVR in physiological conditions, its rate dependence, and the factors contributing to increased BVR in pathologies remain incompletely understood. Here, we employed computer modeling to provide novel insights into the subcellular mechanisms of BVR under physiological conditions and during simulated drug-induced repolarization prolongation, mimicking long-QT syndromes type 1, 2, and 3. We developed stochastic implementations of 13 major ionic currents and fluxes in a model of canine ventricular-myocyte electrophysiology. Combined stochastic gating of these components resulted in short- and long-term variability, consistent with experimental data from isolated canine ventricular myocytes. The model indicated that the magnitude of stochastic fluctuations is rate dependent due to the rate dependence of action-potential (AP) duration (APD). This process (the “active” component) and the intrinsic nonlinear relationship between membrane current and APD (“intrinsic component”) contribute to the rate dependence of BVR. We identified a major role in physiological BVR for stochastic gating of the persistent Na+ current (INa) and rapidly activating delayed-rectifier K+ current (IKr). Inhibition of IKr or augmentation of INa significantly increased BVR, whereas subsequent β-adrenergic receptor stimulation reduced it, similar to experimental findings in isolated myocytes. In contrast, β-adrenergic stimulation increased BVR in simulated long-QT syndrome type 1. In addition to stochastic channel gating, AP morphology, APD, and beat-to-beat variations in Ca2+ were found to modulate single-cell BVR. Cell-to-cell coupling decreased BVR and this was more pronounced when a model cell with increased BVR was coupled to a model cell with normal BVR. In conclusion, our results provide new insights into the ionic mechanisms underlying BVR and suggest that BVR reflects multiple potentially proarrhythmic parameters, including increased ion-channel stochasticity, prolonged APD, and abnormal Ca2+ handling.

Diastolic Spontaneous Calcium Release From the Sarcoplasmic Reticulum Increases Beat-to-Beat Variability of Repolarization in Canine Ventricular Myocytes After β-Adrenergic Stimulation

Rationale: Spontaneous Ca2+ release (SCR) from the sarcoplasmic reticulum can cause delayed afterdepolarizations and triggered activity, contributing to arrhythmogenesis during &bgr;-adrenergic stimulation. Excessive beat-to-beat variability of repolarization duration (BVR) is a proarrhythmic marker. Previous research has shown that BVR is increased during intense &bgr;-adrenergic stimulation, leading to SCR. Objective: We aimed to determine ionic mechanisms controlling BVR under these conditions. Methods and Results: Membrane potentials and cell shortening or Ca2+ transients were recorded from isolated canine left ventricular myocytes in the presence of isoproterenol. Action-potential (AP) durations after delayed afterdepolarizations were significantly prolonged. Addition of slowly activating delayed rectifier K+ current (IKs) blockade led to further AP prolongation after SCR, and this strongly correlated with exaggerated BVR. Suppressing SCR via inhibition of ryanodine receptors, Ca2+/calmodulin-dependent protein kinase II inhibition, or by using Mg2+ or flecainide eliminated delayed afterdepolarizations and decreased BVR independent of effects on AP duration. Computational analyses and voltage-clamp experiments measuring L-type Ca2+ current (ICaL) with and without previous SCR indicated that ICaL was increased during Ca2+-induced Ca2+ release after SCR, and this contributes to AP prolongation. Prolongation of QT, Tpeak-Tend intervals, and left ventricular monophasic AP duration of beats after aftercontractions occurred before torsades de pointes in an in vivo dog model of drug-induced long-QT1 syndrome. Conclusions: SCR contributes to increased BVR by interspersed prolongation of AP duration, which is exacerbated during IKs blockade. Attenuation of Ca2+-induced Ca2+ release by SCR underlies AP prolongation via increased ICaL. These data provide novel insights into arrhythmogenic mechanisms during &bgr;-adrenergic stimulation besides triggered activity and illustrate the importance of IKs function in preventing excessive BVR.

Dominant-Negative Control of cAMP-Dependent IKs Upregulation in Human Long-QT Syndrome Type 1

Rationale: The mutation A341V in the S6 transmembrane segment of KCNQ1, the &agr;-subunit of the slowly activating delayed-rectifier K+ (IKs) channel, predisposes to a severe long-QT1 syndrome with sympathetic-triggered ventricular tachyarrhythmias and sudden cardiac death. Objective: Several genetic risk modifiers have been identified in A341V patients, but the molecular mechanisms underlying the pronounced repolarization phenotype, particularly during &bgr;-adrenergic receptor stimulation, remain unclear. We aimed to elucidate these mechanisms and provide new insights into control of cAMP-dependent modulation of IKs. Methods and Results: We characterized the effects of A341V on the IKs macromolecular channel complex in transfected Chinese hamster ovary cells and found a dominant-negative suppression of cAMP-dependent Yotiao-mediated IKs upregulation on top of a dominant-negative reduction in basal current. Phosphomimetic substitution of the N-terminal position S27 with aspartic acid rescued this loss of upregulation. Western blot analysis showed reduced phosphorylation of KCNQ1 at S27, even for heterozygous A341V, suggesting that phosphorylation defects in some (mutant) KCNQ1 subunits can completely suppress IKs upregulation. Functional analyses of heterozygous KCNQ1 WT:G589D and heterozygous KCNQ1 WT:S27A, a phosphorylation-inert substitution, also showed such suppression. Immunoprecipitation of Yotiao with KCNQ1-A341V (in the presence of KCNE1) was not different from wild-type. Conclusions: Our results indicate the involvement of the KCNQ1-S6 region at/or around A341 in cAMP-dependent stimulation of IKs, a process that is under strong dominant-negative control, suggesting that tetrameric KCNQ1 phosphorylation is required. Specific long-QT1 mutations, including heterozygous A341V, disable this regulation.

New directions in antiarrhythmic drug therapy for atrial fibrillation

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia and has a significant impact on morbidity and mortality. Current antiarrhythmic drugs for AF suffer from limited safety and efficacy, probably because they were not designed based on specific pathological mechanisms. Recent research has provided important insights into the mechanisms contributing to AF and highlighted several potential novel antiarrhythmic strategies. In this review, we highlight the main pathological mechanisms of AF, discuss traditional and novel aspects of atrial antiarrhythmic drugs in relation to these pathological mechanisms, and present potential novel therapeutic approaches including structure-based modulation of atrial-specific cardiac ion channels, restoring abnormal Ca(2+) handling in AF and targeting atrial remodeling.