Increase In Action Potential Duration Research Articles

CaMKII-dependent late Na+ current increases electrical dispersion and arrhythmia in ischemia-reperfusion.

The mechanisms underlying Ca2+/calmodulin-dependent protein kinase II (CaMKII)-induced arrhythmias in ischemia-reperfusion (I/R) are not fully understood. We tested the hypothesis that CaMKII increases late Na+ current ( INa,L) via phosphorylation of Nav1.5 at Ser571 during I/R, thereby increasing arrhythmia susceptibility. To test our hypothesis, we studied isolated, Langendorff-perfused hearts from wild-type (WT) mice and mice expressing Nav channel variants Nav1.5-Ser571E (S571E) and Nav1.5-Ser571A (S571A). WT hearts showed a significant increase in the levels of phosphorylated CaMKII and Nav1.5 at Ser571 [p-Nav1.5(S571)] after 15 min of global ischemia (just before the onset of reperfusion). Optical mapping experiments revealed an increase in action potential duration (APD) and APD dispersion without changes in conduction velocity during I/R in WT and S571E compared with S571A hearts. At the same time, WT and S571E hearts showed an increase in spontaneous arrhythmia events (e.g., premature ventricular contractions) and an increase in the inducibility of reentrant arrhythmias during reperfusion. Pretreatment of WT hearts with the Na+ channel blocker mexiletine (10 μM) normalized APD dispersion and reduced arrhythmia susceptibility during I/R. We conclude that CaMKII-dependent phosphorylation of Nav1.5 is a crucial driver for increased INa,L, arrhythmia triggers, and substrate during I/R. Selective targeting of this CaMKII-dependent pathway may have therapeutic potential for reducing arrhythmias in the setting of I/R. NEW & NOTEWORTHY Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of Nav1.5 at Ser571 leads to a prolongation of action potential duration (APD), increased APD dispersion, and increased arrhythmia susceptibility after ischemia-reperfusion in isolated mouse hearts. Genetic ablation of the CaMKII-dependent phosphorylation site Ser571 on Nav1.5 or low-dose mexiletine (to inhibit late Na+ current) reduced APD dispersion, arrhythmia triggers, and ventricular tachycardia inducibility.

The influences of the M2R-GIRK4-RGS6 dependent parasympathetic pathway on electrophysiological properties of the mouse heart.

A large body of work has established the prominent roles of the atrial M2R-IKACh signaling pathway, and the negative regulatory protein RGS6, in modulating critical aspects of parasympathetic influence on cardiac function, including pace-making, heart rate (HR) variability (HRV), and atrial arrhythmogenesis. Despite increasing evidence of its innervation of the ventricles, and the expression of M2R, IKACh channel subunits, and RGS6 in ventricle, the effects of parasympathetic modulation on ventricular electrophysiology are less clear. The main objective of our study was to investigate the contribution of M2R-IKACh signaling pathway elements in murine ventricular electrophysiology, using in-vivo ECG measurements, isolated whole-heart optical mapping and constitutive knockout mice lacking IKACh (Girk4–/–) or RGS6 (Rgs6-/-). Consistent with previous findings, mice lacking GIRK4 exhibited diminished HR and HRV responses to the cholinergic agonist carbachol (CCh), and resistance to CCh-induced arrhythmic episodes. In line with its role as a negative regulator of atrial M2R-IKACh signaling, loss of RGS6 correlated with a mild resting bradycardia, enhanced HR and HRV responses to CCh, and increased propensity for arrhythmic episodes. Interestingly, ventricles from mice lacking GIRK4 or RGS6 both exhibited increased action potential duration (APD) at baseline, and APD was prolonged by CCh across all genotypes. Similarly, CCh significantly increased the slope of APD restitution in all genotypes. There was no impact of genotype or CCh on either conduction velocity or heterogeneity. Our data suggests that altered parasympathetic signaling through the M2R-IKACh pathway can affect ventricular electrophysiological properties distinct from its influence on atrial physiology.

Mitochondrial Ca2+ Influx Contributes to Arrhythmic Risk in Nonischemic Cardiomyopathy.

BackgroundHeart failure (HF) is associated with increased arrhythmia risk and triggered activity. Abnormal Ca2+ handling is thought to underlie triggered activity, and mitochondria participate in Ca2+ homeostasis.Methods and ResultsA model of nonischemic HF was induced in C57BL/6 mice by hypertension. Computer simulations were performed using a mouse ventricular myocyte model of HF. Isoproterenol‐induced premature ventricular contractions and ventricular fibrillation were more prevalent in nonischemic HF mice than sham controls. Isolated myopathic myocytes showed decreased cytoplasmic Ca2+ transients, increased mitochondrial Ca2+ transients, and increased action potential duration at 90% repolarization. The alteration of action potential duration at 90% repolarization was consistent with in vivo corrected QT prolongation and could be explained by augmented L‐type Ca2+ currents, increased Na+‐Ca2+ exchange currents, and decreased total K+ currents. Of myopathic ventricular myocytes, 66% showed early afterdepolarizations (EADs) compared with 17% of sham myocytes (P<0.05). Intracellular application of 1 μmol/L Ru360, a mitochondrial Ca2+ uniporter–specific antagonist, could reduce mitochondrial Ca2+ transients, decrease action potential duration at 90% repolarization, and ameliorate EADs. Furthermore, genetic knockdown of mitochondrial Ca2+ uniporters inhibited mitochondrial Ca2+ uptake, reduced Na+‐Ca2+ exchange currents, decreased action potential duration at 90% repolarization, suppressed EADs, and reduced ventricular fibrillation in nonischemic HF mice. Computer simulations showed that EADs promoted by HF remodeling could be abolished by blocking either the mitochondrial Ca2+ uniporter or the L‐type Ca2+ current, consistent with the experimental observations.ConclusionsMitochondrial Ca2+ handling plays an important role in EADs seen with nonischemic cardiomyopathy and may represent a therapeutic target to reduce arrhythmic risk in this condition.

ESLICARBAZEPINE ACETATE IS ANTIEPILEPTOGENIC IN THE LATRUNCULIN A MOUSE MODEL OF PARTIAL SEIZURES

Although there are many drugs available which have powerful anticonvulsant activity, there is still a high unmet need for antiepileptogenic compounds that prevent the development of chronic epilepsy. Eslicarbazepine acetate (ESL) is a once‐daily (QD) antiepileptic drug (AED) that is converted extensively to eslicarbazepine (a voltage‐gated sodium channel and T‐type calcium channel blocker) after oral administration.1 ESL is a potent anticonvulsant in animal models. Latrunculin A is an actin depolymerization agent that promotes neurotransmitter release in hippocampal synapses and induces multiple action potential firing and increases action potential duration. Latrunculin A hippocampal microperfusion in freely moving mice induces acute epileptic seizures, followed by the occurrence of chronic sporadic spontaneous seizures, in association with changes in extracellular concentrations of excitatory and inhibitory amino acids.2 We have examined ESL for its potential antiepileptogenic activity in preventing the development of chronic partial seizures induced by latrunculin A microperfusion in the mouse hippocampus.Hippocampi of freely‐moving C57BL/6J male mice were continuously perfused with latrunculin A (8 μg/ml, 1 μl/min, 8h/day) for 72 hours. Sham animals received injections of artificial CSF. Forty‐eight‐hours after intrahippocampal latrunculin A infusion, mice were administered either vehicle, 75 mg/kg or 150 mg/kg ESL p.o. once‐daily for three consecutive days. CSF‐infused mice received vehicle. Body temperature and home‐cage locomotor behaviour were assessed four weeks after the end of ESL treatment. Cortical EEG monitoring by telemetry was performed once per week after the end of ESL treatment over a period of six weeks. Data is presented as means ± standard error of the mean (SEM). Data was analyzed using One‐ or Two‐way ANOVA followed by Holm‐Sidak's multiple comparisons test, as appropriate; planned comparison were performed between sham or vehicle (control) and ESL treated groups.Latrunculin A microperfusion in the mouse hippocampus induced electrographic seizures and discharges in the mouse. EEG monitoring showed that transitory ESL treatment within the epileptogenic period caused a sustained significant decrease in the frequency (42 and 74% decrease for 75 and 150 mg/kg ESL versus vehicle at week 4, respectively; 62 and 72% decrease for 75 and 150 mg/kg ESL versus vehicle at week 6, respectively) of epileptiform discharges. There were no significant differences between the two doses of ESL used. Latrunculin A microperfusion and transitory ESL treatment induced no changes in body temperature and locomotor activity, as assessed on week 4 after latrunculin A administration.Transitory oral ESL treatment attenuated sporadic spontaneous electrographic seizures and discharges induced by latrunculin A microperfusion in the mouse hippocampus, supporting a possible anti‐epileptogenic effect of ESL.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cardiac Kir2.1 and NaV1.5 Channels Traffic Together to the Sarcolemma to Control Excitability.

In cardiomyocytes, NaV1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. The objective of this study was to test whether NaV1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1Δ314-315 or Kir2.1R44A/R46A with wild-type (WT) NaV1.5WT in heterologous cells reduced inward sodium current compared with NaV1.5WT alone or coexpressed with Kir2.1WT. In cell surface biotinylation experiments, expression of Kir2.1Δ314-315 reduced NaV1.5 channel surface expression. Glycosylation analysis suggested that NaV1.5WT and Kir2.1WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of NaV1.5WT, and vice versa, whereas coexpression with Kir2.1Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1Δ314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with NaV1.5WT and Kir2.1WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1WT, NaV1.5WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that NaV1.5WT channels interact with AP1 through the NaV1.5Y1810 residue, suggesting that, like for Kir2.1WT, AP1 can mark NaV1.5 channels for incorporation into clathrin-coated vesicles at the trans-Golgi. Silencing the AP1 ϒ-adaptin subunit in human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. The NaV1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of NaV1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.

Mitochondrial Ca2+ Influx Contributes to Arrhythmic Risk in Nonischemic Cardiomyopathy

Introduction: It had been reported that mitochondrial Ca2+ was overloaded in ischemia-reperfusion injury. Inhibition of mitochondrial Ca2+ uniporter (MCU) prevented cell death. In this study, pharmacological block or genetic knockdown MCU inhibited mitochondrial Ca2+ uptake, depressed early afterdepolarizations (EADs) and reduced ventricular fibrillation in nonischemic heart failure (NI-HF) mice. Methods: A model of NI-HF was induced in CD1 mice by hypertension. Echocardiography, patch-clamp (and Rhod-2 fluorescence density recorded synchronously), telemetry monitoring, real-time PCR and Western blotting were employed in this study. Computer simulations were carried out. Results: Echocardiography showed an impairment of systolic function as the average EF value decreased from 63 ± 2% to 51 ± 3% in NI-HF mice (p<0.05). Isolated myopathic myocytes showed decreased cytoplasmic Ca2+ transients, increased mitochondrial Ca2+ transients, and increased action potential duration at 90% (APD90) repolarization. The alteration of APD90 was consistent with in vivo QTc prolongation and could be explained by augmented L-type Ca2+ currents, increased NCX and decreased total K+ currents. Sixty-six percent of myopathic ventricular myocytes showed EADs compared with 17% of sham myocytes (p<0.05). Intracellular application of 1 µM Ru360 could reduce mitochondrial Ca2+ transients, decrease APD90 and ameliorate EADs. Furthermore, genetic knockdown of MCU inhibited mitochondrial Ca2+ uptake, reduced NCX, decreased APD90, depressed EADs, and reduced ventricular fibrillation in NI-HF mice. Inhibiting mitochondrial NCX slowed mitochondrial Ca2+ uptake, decreased APD90, and depressed EADs. Computer simulations showed that EADs were promoted by heart failure remodeling, which were abolished by blocking either the MCU or the L-type Ca2+ current. Simulations revealed that EADs were promoted via a positive feedback loop among APD, SR Ca2+, and mitochondrial Ca2+, resulting in an all-or-none behavior. Conclusions: Mitochondrial Ca2+ handling plays an important role in EADs seen with chronic and moderate nonischemic cardiomyopathy.

A model for cooperative gating of L-type Ca2+ channels and its effects on cardiac alternans dynamics.

In ventricular myocytes, membrane depolarization during the action potential (AP) causes synchronous activation of multiple L-type CaV1.2 channels (LTCCs), which trigger the release of calcium (Ca2+) from the sarcoplasmic reticulum (SR). This results in an increase in intracellular Ca2+ (Cai) that initiates contraction. During pulsus alternans, cardiac contraction is unstable, going from weak to strong in successive beats despite a constant heart rate. These cardiac alternans can be caused by the instability of membrane potential (Vm) due to steep AP duration (APD) restitution (Vm-driven alternans), instability of Cai cycling (Ca2+-driven alternans), or both, and may be modulated by functional coupling between clustered CaV1.2 (e.g. cooperative gating). Here, mathematical analysis and computational models were used to determine how changes in the strength of cooperative gating between LTCCs may impact membrane voltage and intracellular Ca2+ dynamics in the heart. We found that increasing the degree of coupling between LTCCs increases the amplitude of Ca2+ currents (ICaL) and prolongs AP duration (APD). Increased AP duration is known to promote cardiac alternans, a potentially arrhythmogenic substrate. In addition, our analysis shows that increasing the strength of cooperative activation of LTCCs makes the coupling of Ca2+ on the membrane voltage (Cai→Vm coupling) more positive and destabilizes the Vm-Cai dynamics for Vm-driven alternans and Cai-driven alternans, but not for quasiperiodic oscillation. These results suggest that cooperative gating of LTCCs may have a major impact on cardiac excitation-contraction coupling, not only by prolonging APD, but also by altering Cai→Vm coupling and potentially promoting cardiac arrhythmias.

Optogenetic sensors in the zebrafish heart: a novel in vivo electrophysiological tool to study cardiac arrhythmogenesis

Cardiac arrhythmias are among the most challenging human disorders to diagnose and treat due to their complex underlying pathophysiology. Suitable experimental animal models are needed to study the mechanisms causative for cardiac arrhythmogenesis. To enable in vivo analysis of cardiac cellular electrophysiology with a high spatial and temporal resolution, we generated and carefully validated two zebrafish models, one expressing an optogenetic voltage indicator (chimeric VSFP-butterfly CY) and the other a genetically encoded calcium indicator (GCaMP6f) in the heart.Methods: High-speed epifluorescence microscopy was used to image chimeric VSFP-butterfly CY and GCaMP6f in the embryonic zebrafish heart, providing information about the spatiotemporal patterning of electrical activation, action potential configuration and intracellular Ca2+ dynamics. Plotting VSFP or GCaMP6f signals on a line along the myocardial wall over time facilitated the visualization and analysis of electrical impulse propagation throughout the heart. Administration of drugs targeting the sympathetic nervous system or cardiac ion channels was used to validate sensitivity and kinetics of both zebrafish sensor lines. Using the same microscope setup, we imaged transparent juvenile casper fish expressing GCaMP6f, demonstrating the feasibility of imaging cardiac optogenetic sensors at later stages of development.Results: Isoproterenol slightly increased heart rate, diastolic Ca2+ levels and Ca2+ transient amplitudes, whereas propranolol caused a profound decrease in heart rate and Ca2+ transient parameters in VSFP-Butterfly and GCaMP6f embryonic fish. Ikr blocker E-4031 decreased heart rate and increased action potential duration in VSFP-Butterfly fish. ICa,L blocker nifedipine caused total blockade of Ca2+ transients in GCaMP6f fish and a reduced heart rate, altered ventricular action potential duration and disrupted atrial-ventricular electrical conduction in VSFP-Butterfly fish. Imaging of juvenile animals demonstrated the possibility of employing an older zebrafish model for in vivo cardiac electrophysiology studies. We observed differences in atrial and ventricular Ca2+ recovery dynamics between 3 dpf and 14 dpf casper fish, but not in Ca2+ upstroke dynamics.Conclusion: By introducing the optogenetic sensors chimeric VSFP-butterfly CY and GCaMP6f into the zebrafish we successfully generated an in vivo cellular electrophysiological readout tool for the zebrafish heart. Complementary use of both sensor lines demonstrated the ability to study heart rate, cardiac action potential configuration, spatiotemporal patterning of electrical activation and intracellular Ca2+ homeostasis in embryonic zebrafish. In addition, we demonstrated the first successful use of an optogenetic sensor to study cardiac function in older zebrafish. These models present a promising new research tool to study the underlying mechanisms of cardiac arrhythmogenesis.

Whole exome sequencing identifies a KCNJ12 mutation as a cause of familial dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation, and is associated with systolic dysfunction and increased action potential duration. Approximately 50% of DCM cases are caused by inherited gene mutations with genetic and phenotypic heterogeneity. Next generation sequencing may be useful in screening unknown mutations in such cases.A family was identified with DCM, in which the affected family members developed heart failure, arrhythmia, and sudden death. Probands and 4 affected family members underwent whole exome sequencing (WES), bioinformatics methods, and gene annotation to identify potentially causative variants. The Sanger sequencing method was used to verify the candidate mutation.WES yielded 2,238,831 variations. KCNJ12 (p.Glu334del) was identified as a candidate mutation, and the heterozygous mutation was verified by Sanger sequencing.Our study emphasizes the application of WES in identifying causative mutations in DCM. This report is the first to describe the KCNJ12 gene as a cause of DCM in patients.

Ca2 +-activated Cl− current is antiarrhythmic by reducing both spatial and temporal heterogeneity of cardiac repolarization

The role of Ca2+-activated Cl− current (ICl(Ca)) in cardiac arrhythmias is still controversial. It can generate delayed afterdepolarizations in Ca2+-overloaded cells while in other studies incidence of early afterdepolarization (EAD) was reduced by ICl(Ca). Therefore our goal was to examine the role of ICl(Ca) in spatial and temporal heterogeneity of cardiac repolarization and EAD formation.Experiments were performed on isolated canine cardiomyocytes originating from various regions of the left ventricle; subepicardial, midmyocardial and subendocardial cells, as well as apical and basal cells of the midmyocardium. ICl(Ca) was blocked by 0.5mmol/L 9-anthracene carboxylic acid (9-AC). Action potential (AP) changes were tested with sharp microelectrode recording. Whole-cell 9-AC-sensitive current was measured with either square pulse voltage-clamp or AP voltage-clamp (APVC). Protein expression of TMEM16A and Bestrophin-3, ion channel proteins mediating ICl(Ca), was detected by Western blot.9-AC reduced phase-1 repolarization in every tested cell. 9-AC also increased AP duration in a reverse rate-dependent manner in all cell types except for subepicardial cells. Neither ICl(Ca) density recorded with square pulses nor the normalized expressions of TMEM16A and Bestrophin-3 proteins differed significantly among the examined groups of cells. The early outward component of ICl(Ca) was significantly larger in subepicardial than in subendocardial cells in APVC setting. Applying a typical subepicardial AP as a command pulse resulted in a significantly larger early outward component in both subepicardial and subendocardial cells, compared to experiments when a typical subendocardial AP was applied.Inhibiting ICl(Ca) by 9-AC generated EADs at low stimulation rates and their incidence increased upon beta-adrenergic stimulation. 9-AC increased the short-term variability of repolarization also.We suggest a protective role for ICl(Ca) against risk of arrhythmias by reducing spatial and temporal heterogeneity of cardiac repolarization and EAD formation.

Additive Proarrhythmic Effect of Combined Treatment with QT-Prolonging Agents.

Drug combinations may elevate the risk of proarrhythmia. The aim of the present study was to investigate whether combinations of non-cardiovascular agents induce an additive increase in the proarrhythmic risk. In 12 female rabbit hearts, a drug combination of cotrimoxazole (300µM), ondansetron (5µM) and domperidone (1µM) was infused after obtaining baseline data. In another 13 hearts, a combination of cotrimoxazole (300µM), ondansetron (5µM) and erythromycin (300µM) was infused. Monophasic action potentials and ECG displayed a significant QT prolongation in all groups. This was accompanied by a significant increase in action potential duration. Of note, addition of each drug resulted in a further increase in the QT interval. Furthermore, a significant elevation of spatial dispersion of repolarization was observed. Lowering of potassium concentration in bradycardic AV-blocked hearts provoked early afterdepolarizations and torsade de pointes (TDP) in both study groups. Under baseline conditions, no episodes of TDP recorded. After administration of the first agent, TDP occurred in 5 of 12 hearts (37 episodes) and 5 of 13 hearts (26 episodes), respectively. After additional infusion of the second drug, TDP were recorded in 7 of 12 hearts (55 episodes) and 8 of 13 hearts (111 episodes). After additional infusion of the third drug, TDP occurred in 11 of 12 hearts (118 episodes) and 9 of 13 hearts (88 episodes). Combined treatment with several non-cardiovascular QT-prolonging agents resulted in a remarkable occurrence of proarrhythmia. An additive and significant prolongation of cardiac repolarization combined with an increased spatial dispersion of repolarization represents the underlying electrophysiological mechanism.

Effects of new antiarrhythmic agent SS-68 on excitation conduction, electrical activity in Purkinje fibers and pulmonary veins: Assessment of safety and side effects risk

The compound SS-68 has been selected among numerous new derivatives of indole and demonstrated antiarrhythmic effects in animal models. The present study concerns several aspects of SS-68 safety and efficacy as a potential antiarrhythmic drug. The first estimation of atrioventricular conduction in mammalian heart under SS-68 has been carried out; effects of SS-68 in Purkinje fibers and myocardium of pulmonary veins have been investigated.The drug weakly affects cardiac atrioventricular conduction: only high concentrations of SS-68 (≥10 μmol/L) significantly decrease this parameter. Also, the drug weakly affects Purkinje fibers automaticity, but effectively alters action potential waveform in Purkinje fibers in a concentration-dependent manner. SS-68 (0.1–100 μmol/L) failed to induce any early or delayed afterdepolarizations in Purkinje fibers both in basal conditions and under provocation of proarrhythmic activity by norepinephrine (NE). Moreover, 10 μmol/L SS-68 suppressed NE-induced extra-beats and rapid firing in Purkinje fibers. In pulmonary veins only high concentrations of SS-68 significantly increased action potential duration, while lower concentrations (0.1–1 μmol/L) were ineffective. Also, 0.1–100 μmol/L SS-68 was unable to elicit arrhythmogenic alternations of action potential waveform in pulmonary veins.In conclusion, SS-68 has no proarrhythmic effects, such as afterdepolarizations or abnormal automaticity in used experimental models.

Severe Proarrhythmic Potential of the Antiemetic Agents Ondansetron and Domperidone.

The potential of ondansetron and domperidone, both clinically established antiemetic agents, to increase the QT-interval has been described in several case reports. Therefore, the aim of the present study was to investigate whether these drugs may provoke polymorphic ventricular tachycardia in a sensitive experimental model of drug-induced proarrhythmia. In 10 female rabbits, ondansetron (1, 5 and 10µM, n=10) or domperidone (0.5, 1 and 2µM, n=8) was infused after obtaining baseline data. Eight endo- and epicardial monophasic action potentials and a simultaneously recorded 12-lead ECG reproduced the clinically observed QT-prolongation (ondansetron: 1µM:+17ms, 5µM:+41ms, 10µM:+78ms, p<0.01; domperidone: 0.5µM:+57ms, 1µM:+79ms, 2µM:+99ms, p<0.01). This was accompanied by a significant increase in action potential duration at 90% of repolarization. Administration of both agents also increased dispersion of repolarization (ondansetron: 1µM:+12ms, 5µM:+17ms; 10µM:+18ms, p<0.05; domperidone: 0.5µM:+19ms, 1µM:+27ms; 2µM:+23ms p<0.05). Lowering of potassium concentration in bradycardic AV-blocked hearts provoked early afterdepolarizations (EADs) in 9 of 10 ondansetron-treated hearts and induced polymorphic ventricular tachycardia (VT) resembling torsade de pointes in 7 of 10 ondansetron-treated hearts (86 episodes). Under the influence of domperidone, EAD and polymorphic VT occurred in 7 of 8 hearts (131 episodes). In the present study, both ondansetron and domperidone demonstrated a severe proarrhythmic potential. A significant prolongation of cardiac repolarization as well as a marked increase in spatial dispersion of repolarization represents the underlying electrophysiologic mechanisms. These results imply that application of ondansetron should be handled carefully. For regular administration, ECG monitoring should be mandatory.

Electrophysiological Effects of Triggered Calcium Waves on the Action Potential in Left Atrial Myocytes from Normal and Failing Dog Heart

We previously reported that triggered Ca waves (TCWs) occur in dog atrial myocytes during rapid pacing. The goal of this study was to investigate the effect of TCWs on APs during rapid pacing in left atrial myocytes from both normal and failing hearts (rapid RV pacing model). Intracellular Ca and AP recordings were made in atrial myocytes isolated from normal and failing (HF) dog hearts loaded with Ca-sensitive fluorescent dyes and stimulated at increasingly rapid rates (1-5Hz). Both normal and HF atrial myocytes developed TCWs which increased action potential duration (APD) and reduced maximum diastolic potential (MDP) throughout the duration of each TCW. Depending on the timing of the TCW, both MDP and APD could be affected during a single or multiple cycles. TCWs developed at slower rates in HF than in normals so changes in AP and MDP also occurred at slower rates making TCWs of potentially greater importance to arrhythmogenesis in HF. A subset of TCWs failed to propagate along the cell length but were short enough to occur during only 1-2 APs. These events (triggered Ca flares, TCFs) produce the same overall electrophysiological effects as TCWs that propagate along the cell length. In summary, TCFs and TCWs cause APD prolongation and cell depolarization in normal and diseased atrial myocytes probably by inducing inward Na+-Ca2+ exchange current in response to these Ca release events. This depolarization and slowing of repolarization is likely to establish repolarization gradients and cause reentry thus promoting atrial arrhythmogenesis such as atrial fibrillation.

Conditional knockout of Fgf13 in murine hearts increases arrhythmia susceptibility and reveals novel ion channel modulatory roles

The intracellular fibroblast growth factors (iFGF/FHFs) bind directly to cardiac voltage gated Na+ channels, and modulate their function. Mutations that affect iFGF/FHF-Na+ channel interaction are associated with arrhythmia syndromes. Although suspected to modulate other ionic currents, such as Ca2+ channels based on acute knockdown experiments in isolated cardiomyocytes, the in vivo consequences of iFGF/FHF gene ablation on cardiac electrical activity are still unknown. We generated inducible, cardiomyocyte-restricted Fgf13 knockout mice to determine the resultant effects of Fgf13 gene ablation. Patch clamp recordings from ventricular myocytes isolated from Fgf13 knockout mice showed a ~25% reduction in peak Na+ channel current density and a hyperpolarizing shift in steady-state inactivation. Electrocardiograms on Fgf13 knockout mice showed a prolonged QRS duration. The Na+ channel blocker flecainide further prolonged QRS duration and triggered ventricular tachyarrhythmias only in Fgf13 knockout mice, suggesting that arrhythmia vulnerability resulted, at least in part, from a loss of functioning Na+ channels. Consistent with these effects on Na+ channels, action potentials in Fgf13 knockout mice, compared to Cre control mice, exhibited slower upstrokes and reduced amplitude, but unexpectedly had longer durations. We investigated candidate sources of the prolonged action potential durations in myocytes from Fgf13 knockout mice and found a reduction of the transient outward K+ current (Ito). Fgf13 knockout did not alter whole-cell protein levels of Kv4.2 and Kv4.3, the Ito pore-forming subunits, but did decrease Kv4.2 and Kv4.3 at the sarcolemma. No changes were seen in the sustained outward K+ current or voltage-gated Ca2+ current, other candidate contributors to the increased action potential duration. These results implicate that FGF13 is a critical cardiac Na+ channel modulator and Fgf13 knockout mice have increased arrhythmia susceptibility in the setting of Na+ channel blockade. The unanticipated effect on Ito revealed new FGF13 properties and the unexpected lack of an effect on voltage-gated Ca2+ channels highlight potential compensatory changes in vivo not readily revealed with acute Fgf13 knockdown in cultured cardiomyocytes.

Terminating spiral wave and spatiotemporal chaos in cardiac tissues by using late sodium current

Most Na+ channels open transiently upon depolarization of cardiac cell membrane and then are quickly inactivated. However, some Na+ channels remain active, which generate the late sodium current during the action potential plateau. So far, late sodium current has been regarded as a relevant contributor to arrhythmias and its inhibition can suppress re-entrant and multifocal ventricular fibrillation so that its inhibition may become a novel therapeutic strategy to treat cardiac arrhythmias in the future. Therefore, how to inhibit late sodium current has received special attention. Since both the late sodium current and defibrillation shocks can lead to the increase of action potential duration, the late sodium current can be used to terminate ventricular fibrillation. However, the suppression of spiral wave and spatiotemporal chaos in cardiac tissues via late sodium current has been neglected. In this paper, we use the model of human heart to study the suppression of spiral wave and spatiotemporal chaos in two-dimensional cardiac tissue by generating late sodium current. We suggest that such a control strategy to induce late sodium current. The slow inactivation gate of sodium channel is clamped to 0.7 while the threshold voltage of corresponding fast inactivation gate is real-timely modulated. We first reduce the threshold voltage from 71.55 mV to 50.55 mV within the time interval T1, and then increase it from 50.55 mV to 71.55 mV within the time interval T2. When the threshold voltage returns to 71.55 mV, the changes of the relevant inactivation gates of sodium channel go back to normal dynamic state. Numerical simulation results show that when the control parameters are properly chosen, the control-induced late sodium current can effectively suppress spiral wave and spatiotemporal chaos even if there are some cardiac cells with spontaneous late sodium current. The advantage of the control scheme is that the control-induced late sodium current is small. The control duration is short because the spiral wave and spatiotemporal chaos disappear mainly due to the conduction obstacle. In a few cases, the spatiotemporal chaos disappears through the transition from spiral wave to target wave. We hope that these results may provide a new strategy to treat heart disease.

The effects of linalool on the excitability of central neurons of snail Caucasotachea atrolabiata

Linalool is a major constituent of the essential oil of several plant species and possesses several biological activities. In this work, we studied the effects of linalool on excitability of central neurons of land snail Caucasotachea atrolabiata and tried to elucidate the underlying mechanisms. The lower concentration of linalool (0.1mM) showed suppressive action on spontaneous activity and pentylenetetrazole-induced epileptiform activity. These effects were associated with elevation of the action potential threshold and reduction of action potential rising phase, supporting the inhibitory action of linalool on Na+ channels. At this concentration it also prolonged the post stimulus inhibitory period that can take part in its antiepileptic effect and apparently results from increased action potential duration and indirect augmentation of Ca2+-activated K+ currents. At higher concentration, however, linalool (0.4mM) increased the neuronal excitability and induced epileptiform activity. The modulatory effects on action potential waveform during preconvulsive period suggest that the recent effect is mainly dependent on the suppression of outward potassium currents underlying repolarization phase and afterhyperpolarization. The linalool-induced epileptiform activity was abolished by Ca2+ channel blockers, nifedipine and nickel chloride, and selective inhibitor of protein kinase C, chelerythrine, suggesting that Ca2+ inward currents and protein kinase C (PKC) activity are required for linalool-induced epileptiform activity.Our results support the antiepileptic activity of linalool at lower dose, but it shows epileptogenic activity when applied directly on snail neurons at higher dose. Linalool may also be a potential therapeutic agent for activating PKC.

Antiarrhythmic effect of vernakalant in an experimental model of Long-QT-syndrome.

The antiarrhythmic drug vernakalant exerts antiarrhythmic effects in atrial fibrillation. Recent experimental data suggest interactions with the late sodium current and antiarrhythmic effects in ventricular arrhythmias. We aimed at investigating whether treatment with vernakalant reduces polymorphic ventricular tachycardia (VT) in an experimental model of Long-QT-syndrome (LQTS). Twenty-nine isolated rabbit hearts were assigned to two groups and treated with erythromycin (300 µM, n = 15) or veratridine (0.5 µM, n = 14) after obtaining baseline data. Thereafter, vernakalant (10 µM) was additionally infused. Infusion of erythromycin or veratridine significantly increased action potential duration (APD90) and QT interval. Erythromycin and veratridine also significantly augmented spatial dispersion of repolarization (erythromycin: +43 ms; veratridine: +55 ms, P < 0.01, respectively) and temporal dispersion of repolarization. After lowering extracellular [K+] in bradycardic hearts, 11 of 15 erythromycin-treated hearts and 4 of 14 veratridine-treated hearts showed early afterdepolarizations and subsequent polymorphic VT. Additional treatment with vernakalant resulted in a significant reduction of spatial dispersion of spatial dispersion in both groups (erythromycin: -32 ms; veratridine: -35 ms, P < 0.05 each) and a stabilization of temporal dispersion. After additional treatment with vernakalant, only 5 of 15 erythromycin-treated hearts (P = 0.07) and 1 of 14 veratridine-treated hearts (P = 0.32) presented polymorphic VT. Vernakalant has antiarrhythmic effects in this experimental model of acquired LQTS. A reduction of spatial dispersion of repolarization and a stabilization of temporal dispersion in hearts showing polymorphic VT represent the major underlying electrophysiological mechanisms.

Severe proarrhythmic potential of risperidone compared to quetiapine in an experimental whole-heart model of proarrhythmia

In several case reports, proarrhythmic effects of antipsychotic drugs have been reported. The aim of the present study was to investigate if application of risperidone or quetiapine has the potential to provoke polymorphic ventricular tachycardia in a sensitive model of proarrhythmia. In 24 isolated rabbit hearts, risperidone (5 and 10μM, n=12) or quetiapine (5 and 10μM, n=12) was infused after obtaining baseline data. Eight endocardial and epicardial monophasic action potentials and a simultaneously recorded 12-lead ECG showed a significant QT prolongation after application of risperidone as compared with baseline (5μM: +29ms, 10μM: +35ms, p<0.01) accompanied by an increase of action potential duration. Administration of risperidone also significantly increased spatial dispersion of repolarization (5μM: +16ms, 4μM: +19ms; p<0.05) as well as temporal dispersion of repolarization. Lowering of potassium concentration in bradycardic AV-blocked hearts provoked early afterdepolarizations (EADs) in 8 of 12 hearts and polymorphic ventricular tachycardia resembling torsade de pointes in 6 of 12 hearts (10μM, 49 episodes). The results were compared with hearts treated with quetiapine (5 and 10μM). Quetiapine led to an increase in QT interval (5μM: +10ms; 10μM: +28ms; p<0.05) and a similar increase of APD90. However, treatment with quetiapine did not result in significant alterations of spatial and temporal dispersion of repolarization. No ventricular arrhythmias were observed in this group. In the present study, quetiapine demonstrated a safe electrophysiologic profile despite significant QT prolongation. In contrast, risperidone led to a more marked prolongation of myocardial repolarization combined with a more marked increase of dispersion of repolarization.

Low proarrhythmic potential of citalopram and escitalopram in contrast to haloperidol in an experimental whole-heart model

In several case reports proarrhythmic effects of citalopram and escitalopram have been reported. Systematic analyses on prorarrhythmic effects of these drugs are not yet available.The aim of the present study was to investigate if application of citalopram, escitalopram or haloperidol provokes polymorphic ventricular tachycardia in a sensitive model of proarrhythmia.In isolated rabbit hearts monophasic action potentials and ECG showed a significant QT-prolongation after application of citalopram (2µM: +47ms, 4µM: +56ms, P<0.05) accompanied by an increase of action potential duration (APD) but not dispersion of repolarization. Reduced potassium concentration in bradycardic AV-blocked hearts provoked early afterdepolarizations (EAD) in 2 of 12 hearts but no polymorphic ventricular tachycardia (pVT). Application of escitalopram also increased QT-interval (2µM: +3ms, 4µM: +30ms, P<0.05) and APD without effects on dispersion. 3 of 10 hearts showed EAD and pVT in 2 of 10 hearts (32 episodes).The results were compared to 12 rabbits treated with haloperidol which led to an increase in QT-interval (1µM:+62ms; 2µM:+96ms; P<0.01), APD and dispersion (1µM:+15ms, 2µM:+40ms; P<0.01) and induced EAD in all 12 and pVT in 10 of 12 hearts (152 episodes).Citalopram and escitalopram demonstrated a rather safe electrophysiologic profile despite significant QT prolongation. In contrast, haloperidol led to significant increase of dispersion of repolarization while this parameter remained stable under the influence of citalopram or escitalopram. These results imply that application of citalopram or escitalopram is not as proarrhythmic as some case reports might suggest while haloperidol is torsadogenic.