The mechanism of Atrial Fibrillation (AF) that emerges spontaneously during acute oxidative stress is poorly defined and its drug therapy remains suboptimal. We hypothesized that oxidative activation of Ca-calmodulin dependent protein kinase (CaMKII) promotes Early Afterdepolarization-(EAD)-mediated triggered AF in aged fibrotic atria that is sensitive to late Na current (INa-L) blockade. High-resolution voltage optical mapping of the Left and Right Atrial (LA & RA) epicardial surfaces along with microelectrode recordings were performed in isolated-perfused male Fisher 344 rat hearts in Langendorff setting. Aged atria (23-24 months) manifested 10-fold increase in atrial tissue fibrosis compared to young/adult (2-4 months) atria (P<0001. Spontaneous AF arose in 39 out of 41 of the aged atria but in 0 out of 12 young/adult hearts (P<001) during arterial perfusion of with 0.1 mm of hydrogen peroxide (H2O2). Optical Action Potential (AP) activation maps showed that the AF was initiated by a focal mechanism in the LA suggestive of EAD-mediated triggered activity. Cellular AP recordings with glass microelectrodes from the LA epicardial sites showing focal activity confirmed optical AP recordings that the spontaneous AF was initiated by late phase 3 EAD-mediated triggered activity. Inhibition of CaMKII activity with KN-93 (1 μM) (N=6) or its downstream target, the enhanced INa-L with GS-967 (1 μM), a specific blocker of INa-L (N=6), potently suppressed the AF and prevented its initiation when perfused 15 min prior to H2O2 (n=6). Increased atrial tissue fibrosis combined with acute oxidative activation of CaMK II Initiate AF by EAD-mediated triggered activity. Specific block of the INa-L with GS-967 effectively suppresses the AF. Drug therapy of oxidative AF in humans with traditional antiarrhythmic drugs remains suboptimal; suppressing INa-L offers a potential new strategy for effective suppression of oxidative human AF that remains suboptimal.

Late Na⁺ current (I_NaL) significantly contributes to shaping cardiac action potentials (APs) and increased I_NaL is associated with cardiac arrhythmias. β-adrenergic receptor (βAR) stimulation and its downstream signaling via protein kinase A (PKA) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) pathways are known to regulate I_NaL. However, it remains unclear how each of these pathways regulates I_NaL during the AP under physiological conditions. Here we performed AP-clamp experiments in rabbit ventricular myocytes to delineate the impact of each signaling pathway on I_NaL at different AP phases to understand the arrhythmogenic potential. During the physiological AP (2 Hz, 37 °C) we found that I_NaL had a basal level current independent of PKA, but partially dependent on CaMKII. βAR activation (10 nM isoproterenol, ISO) further enhanced I_NaL via both PKA and CaMKII pathways. However, PKA predominantly increased I_NaL early during the AP plateau, whereas CaMKII mainly increased I_NaL later in the plateau and during rapid repolarization. We also tested the role of key signaling pathways through exchange protein activated by cAMP (Epac), nitric oxide synthase (NOS) and reactive oxygen species (ROS). Direct Epac stimulation enhanced I_NaL similar to the βAR-induced CaMKII effect, while NOS inhibition prevented the βAR-induced CaMKII-dependent I_NaL enhancement. ROS generated by NADPH oxidase 2 (NOX2) also contributed to the ISO-induced I_NaL activation early in the AP. Taken together, our data reveal differential modulations of I_NaL by PKA and CaMKII signaling pathways at different AP phases. This nuanced and comprehensive view on the changes in I_NaL during AP deepens our understanding of the important role of I_NaL in reshaping the cardiac AP and arrhythmogenic potential under elevated sympathetic stimulation, which is relevant for designing therapeutic treatment of arrhythmias under pathological conditions.

It is imperative to develop better approaches to predict how antiarrhythmic drugs with multiple interactions and targets may alter the overall electrical and/or mechanical function of the heart. Safety Pharmacology studies have provided new insights into the multi-target effects of many different classes of drugs and have been aided by the addition of robust new in vitro and in silico technology. The primary focus of Safety Pharmacology studies has been to determine the risk profile of drugs and drug candidates by assessing their effects on repolarization of the cardiac action potential. However, for decades experimental and clinical studies have described substantial and potentially detrimental effects of Na⁺ channel blockers in addition to their well-known conduction slowing effects. One such side effect, associated with administration of some Na⁺ channel blocking drugs is negative inotropy. This reduces the pumping function of the heart, thereby resulting in hypotension. Flecainide is a well-known example of a Na⁺ channel blocking drug, that exhibits strong rate-dependent block of I_Na and may cause negative cardiac inotropy. While the phenomenon of Na⁺ channel suppression and resulting negative inotropy is well described, the mechanism(s) underlying this effect are not. Here, we set out to use a modeling and simulation approach to reveal plausible mechanisms that could explain the negative inotropic effect of flecainide. We utilized the Grandi-Bers model [1] of the cardiac ventricular myocyte because of its robust descriptions of ion homeostasis in order to characterize and resolve the relative effects of QRS widening, flecainide off-target effects and changes in intracellular Ca²⁺ and Na⁺ homeostasis. The results of our investigations and predictions reconcile multiple data sets and illustrate how multiple mechanisms may play a contributing role in the flecainide induced negative cardiac inotropic effect.

Background

Rapid delayed rectifier K⁺ current (I_Kr) and late Na⁺ current (I_NaL) significantly shape the cardiac action potential (AP). Changes in their magnitudes can cause either long or short QT syndromes associated with malignant ventricular arrhythmias and sudden cardiac death.

Methods

Physiological self AP-clamp was used to measure I_NaL and I_Kr during the AP in rabbit and porcine ventricular cardiomyocytes to test our hypothesis that the balance between I_Kr and I_NaL affects repolarization stability in health and disease conditions.

Results

We found comparable amount of net charge carried by I_Kr and I_NaL during the physiological AP, suggesting that outward K⁺ current via I_Kr and inward Na⁺ current via I_NaL are in balance during physiological repolarization. Remarkably, I_Kr and I_NaL integrals in each control myocyte were highly correlated in both healthy rabbit and pig myocytes, despite high overall cell-to-cell variability. This close correlation was lost in heart failure myocytes from both species. Pretreatment with E-4031 to block I_Kr (mimicking long QT syndrome 2) or with sea anemone toxin II to impair Na⁺ channel inactivation (mimicking long QT syndrome 3) prolonged AP duration (APD); however, using GS-967 to inhibit I_NaL sufficiently restored APD to control in both cases. Importantly, I_NaL inhibition significantly reduced the beat-to-beat and short-term variabilities of APD. Moreover, I_NaL inhibition also restored APD and repolarization stability in heart failure. Conversely, pretreatment with GS-967 shortened APD (mimicking short QT syndrome), and E-4031 reverted APD shortening. Furthermore, the amplitude of AP alternans occurring at high pacing frequency was decreased by I_NaL inhibition, increased by I_Kr inhibition, and restored by combined I_NaL and I_Kr inhibitions.

Conclusions

Our data demonstrate that I_Kr and I_NaL are counterbalancing currents during the physiological ventricular AP and their integrals covary in individual myocytes. Targeting these ionic currents to normalize their balance may have significant therapeutic potential in heart diseases with repolarization abnormalities. Visual Overview: A visual overview is available for this article.

Background

The QT interval is a phase of the cardiac cycle that corresponds to action potential duration (APD) including cellular repolarization (T-wave). In both clinical and experimental settings, prolongation of the QT interval of the electrocardiogram (ECG) and related proarrhythmia have been so strongly associated that a prolonged QT interval is largely accepted as surrogate marker for proarrhythmia. Accordingly, drugs that prolong the QT interval are not considered for further preclinical development resulting in removal of many promising drugs from development. While reduction of drug interactions with hERG is an important goal, there are promising means to mitigate hERG block. Here, we examine one possibility and test the hypothesis that selective inhibition of the cardiac late Na current (I_NaL) by the novel compound GS-458967 can suppress proarrhythmic markers.

Methods and results

New experimental data has been used to calibrate I_NaL in the Soltis-Saucerman computationally based model of the rabbit ventricular action potential to study effects of GS-458967 on I_NaL during the rabbit ventricular AP. We have also carried out systematic in silico tests to determine if targeted block of I_NaL would suppress proarrhythmia markers in ventricular myocytes described by TRIaD: Triangulation, Reverse use dependence, beat-to-beat Instability of action potential duration, and temporal and spatial action potential duration Dispersion.

Conclusions

Our computer modeling approach based on experimental data, yields results that suggest that selective inhibition of I_NaL modifies all TRIaD related parameters arising from acquired Long-QT Syndrome, and thereby reduced arrhythmia risk. This study reveals the potential for adjunctive pharmacotherapy via targeted block of I_NaL to mitigate proarrhythmia risk for drugs with significant but unintended off-target hERG blocking effects.

Background

Heart failure (HF) is characterized by electrophysiological remodeling resulting in increased risk of cardiac arrhythmias. Previous reports suggest that elevated inward ionic currents in HF promote action potential (AP) prolongation, increased short-term variability of AP repolarization, and delayed afterdepolarizations. However, the underlying changes in late Na⁺ current (I_NaL), L-type Ca²⁺ current, and NCX (Na⁺/Ca²⁺ exchanger) current are often measured in nonphysiological conditions (square-pulse voltage clamp, slow pacing rates, exogenous Ca²⁺ buffers).

Methods

We measured the major inward currents and their Ca²⁺- and β-adrenergic dependence under physiological AP clamp in rabbit ventricular myocytes in chronic pressure/volume overload-induced HF (versus age-matched control).

Results

AP duration and short-term variability of AP repolarization were increased in HF, and importantly, inhibition of I_NaL decreased both parameters to the control level. I_NaL was slightly increased in HF versus control even when intracellular Ca²⁺ was strongly buffered. But under physiological AP clamp with normal Ca²⁺ cycling, I_NaL was markedly upregulated in HF versus control (dependent largely on CaMKII [Ca²⁺/calmodulin-dependent protein kinase II] activity). β-Adrenergic stimulation (often elevated in HF) further enhanced I_NaL. L-type Ca²⁺ current was decreased in HF when Ca²⁺ was buffered, but CaMKII-mediated Ca²⁺-dependent facilitation upregulated physiological L-type Ca²⁺ current to the control level. Furthermore, L-type Ca²⁺ current response to β-adrenergic stimulation was significantly attenuated in HF. Inward NCX current was upregulated at phase 3 of AP in HF when assessed by combining experimental data and computational modeling.

Conclusions

Our results suggest that CaMKII-dependent upregulation of I_NaL in HF significantly contributes to AP prolongation and increased short-term variability of AP repolarization, which may lead to increased arrhythmia propensity, and is further exacerbated by adrenergic stress.

In February 2014, a group of scientists convened as part of the University of California Davis Cardiovascular Symposium to bring together experimental and mathematical modelling perspectives and discuss points of consensus and controversy on the topic of sodium in the heart. This paper summarizes the topics of presentation and discussion from the symposium, with a focus on the role of aberrant sodium channels and abnormal sodium homeostasis in cardiac arrhythmias and pharmacotherapy from the subcellular scale to the whole heart. Two following papers focus on Na(+) channel structure, function and regulation, and Na(+)/Ca(2+) exchange and Na(+)/K(+) ATPase. The UC Davis Cardiovascular Symposium is a biannual event that aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The focus on Na(+) in the 2014 symposium stemmed from the multitude of recent studies that point to the importance of maintaining Na(+) homeostasis in the heart, as disruption of homeostatic processes are increasingly identified in cardiac disease states. Understanding how disruption in cardiac Na(+)-based processes leads to derangement in multiple cardiac components at the level of the cell and to then connect these perturbations to emergent behaviour in the heart to cause disease is a critical area of research. The ubiquity of disruption of Na(+) channels and Na(+) homeostasis in cardiac disorders of excitability and mechanics emphasizes the importance of a fundamental understanding of the associated mechanisms and disease processes to ultimately reveal new targets for human therapy.

Heart failure (HF) following myocardial infarction (MI) is associated with high incidence of cardiac arrhythmias. Development of therapeutic strategy requires detailed understanding of electrophysiological remodeling. However, changes of ionic currents in ischemic HF remain incompletely understood, especially in translational large-animal models. Here, we systematically measure the major ionic currents in ventricular myocytes from the infarct border and remote zones in a porcine model of post-MI HF. We recorded eight ionic currents during the cell's action potential (AP) under physiologically relevant conditions using ^selfAP-clamp sequential dissection. Compared with healthy controls, HF-remote zone myocytes exhibited increased late Na⁺ current, Ca²⁺-activated K⁺ current, Ca²⁺-activated Cl^- current, decreased rapid delayed rectifier K⁺ current, and altered Na⁺/Ca²⁺ exchange current profile. In HF-border zone myocytes, the above changes also occurred but with additional decrease of L-type Ca²⁺ current, decrease of inward rectifier K⁺ current, and Ca²⁺ release-dependent delayed after-depolarizations. Our data reveal that the changes in any individual current are relatively small, but the integrated impacts shift the balance between the inward and outward currents to shorten AP in the border zone but prolong AP in the remote zone. This differential remodeling in post-MI HF increases the inhomogeneity of AP repolarization, which may enhance the arrhythmogenic substrate. Our comprehensive findings provide a mechanistic framework for understanding why single-channel blockers may fail to suppress arrhythmias, and highlight the need to consider the rich tableau and integration of many ionic currents in designing therapeutic strategies for treating arrhythmias in HF.

This paper is the second of a series of three reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on Na(+) channel function and regulation, Na(+) channel structure and function, and Na(+) channel trafficking, sequestration and complexing.