Prolongation Of Cardiac Action Potential Research Articles

Cardiac action potential prolongation in the absence of NCX1 Na+-dependent inactivation: A mechanistic insight.

The electrogenic Na+-Ca2+ exchanger (NCX1) transports three Na+ into the cell while extruding one Ca2+, thus regulating both cardiac excitability and contractility. In addition to being transported, intracellular Na+ inhibits NCX1 via a mechanism termed Na+-dependent inactivation. The physiological role of this regulation remains unknown. We created a novel mouse line in which the native NCX1 carries mutation K229Q. This modification removes NCX1 Na+-dependent inactivation without affecting other regulatory properties (Matsuoka et al., JPG 109:273-86, 1997; John et al., JGP 150:245-257, 2018). K229Q mice show a prolonged QT interval and action potential (Steccanella et al., Biophys. J. 118, 100a, 2020). To determine the mechanism underlying these altered electrical properties, we recorded NCX1 currents from patch clamped myocytes. Currents were elicited by a voltage ramp protocol and acquired in the absence and presence of intracellular Na+. In the absence of cytosolic Na+ both WT and K229Q exchangers do not undergo Na+-dependent inactivation and thus generated similar current amplitudes (WT = −0.13±0.04 pA/pF, n = 7/5 cells/animals; K229Q = −0.12±0.04, n = 7/3; p = 0.87, measured at −70 mV, mean±SEM). In contrast, in the presence of 25mM Na+, NCX1 currents recorded from K229Q cardiomyocytes were significantly larger than those measured from WT cells (WT = 0.72±0.07 pA/pF, n = 8/4; K229Q = 1.07±0.10, n = 10/6; p = 0.009, measured at 50 mV). This is consistent with increased activity of NCX1-K229Q which lacks Na+-dependent inactivation. L-type Ca2+ current amplitudes (WT = −12.26±1.32 pA/pF, n = 7/5; K229Q = −14.82±2.29, n = 6/3; p = 0.36, measured at 10 mV) and transcript levels of CaV1.2, KV2.1, KV4.2, and Kir2.1 channels were not significantly different between WT and K229Q mice. These findings suggest that action potential prolongation observed in K229Q cardiomyocytes is primarily mediated by NCX1 current enhanced by the lack of Na+-dependent inactivation and that NCX1 Na+-dependent inactivation is an important modulator of cardiac electrical activity.

Dual Mechanisms of Cardiac Action Potential Prolongation by 4-Oxo-Nonenal Increasing the Risk of Arrhythmia; Late Na+ Current Induction and hERG K+ Channel Inhibition.

4-Oxo-nonenal (4-ONE) is an endogenous lipid peroxidation product that is more reactive than 4-hydroxy-nonenal (4-HNE). We previously reported the arrhythmic potential of 4-HNE by suppression of cardiac human Ether-a-go-go Related Gene (hERG) K+ channels with prolonged action potential duration (APD) in cardiomyocytes. Here, we illustrate the higher arrhythmic risk of 4-ONE by modulating the cardiac hNaV1.5 channel currents (INaV). Although the peak amplitude of INaV was not significantly changed by 4-ONE up to 10 μM, the rate of INaV inactivation was slowed, and the late Na+ current (INaL) became larger by 10 μM 4-ONE. The chemical modification of specific residues in hNaV1.5 by 4-ONE was identified using MS-fingerprinting analysis. In addition to the changes in INaV, 4-ONE decreased the delayed rectifier K+ channel currents including the hERG current. The L-type Ca2+ channel current was decreased, whereas its inactivation was slowed by 4-ONE. The APD prolongation by 10 μM of 4-ONE was more prominent than that by 100 μM of 4-HNE. In the computational in silico cardiomyocyte simulation analysis, the changes of INaL by 4-ONE significantly exacerbated the risk of arrhythmia exhibited by the TdP marker, qNet. Our study suggests an arrhythmogenic effect of 4-ONE on cardiac ion channels, especially hNaV1.5.

Annexin A4 N-terminal peptide inhibits adenylyl cyclase 5 and limits β-adrenoceptor-mediated prolongation of cardiac action potential.

Adenylyl cyclases (AC) are essential for the normal and pathophysiological response of many cells. In cardiomyocytes, the predominant AC isoforms are AC5 and AC6. Specific AC5 inhibition was suggested as an option for the treatment of heart failure potentially advantageous over β-blockers. We previously reported an interaction between the calcium-binding protein annexin A4 (ANXA4) and AC5 in human embryonic kidney 293 (HEK293) cells and an inhibition of cyclic adenosine monophosphate (cAMP) production in cardiomyocytes. Here, we investigated whether ANXA4 is able to differentiate between AC5 and AC6. In transfected HEK293 cells, ANXA4 specifically co-immunoprecipitated with AC5 and not with AC6, via its N-terminal domain. Both ANXA4 and a peptide comprising the ANXA4 N-terminal sequence (A4N1-22 ) decreased the cAMP production in AC5 and not in AC6 expressing cells. In line with ACs inhibition, in myocytes from ANXA4-deficient mice, β-adrenoceptor (βAR) stimulation led to a higher increase of the L-type calcium current (ICaL ) and to an excessive action potential duration (APD) prolongation as compared to wild-type cardiomyocytes. This enhanced response was reversed in the presence of A4N1-22 peptide likely via specific AC5 inhibition. We conclude that via the N-terminal domain ANXA4 inhibits AC5 not AC6, and that A4N1-22 as a specific AC5 inhibitor could serve as a novel therapeutic tool for the treatment of AC5-linked diseases.

The KCa2 Channel Inhibitor AP14145, But Not Dofetilide or Ondansetron, Provides Functional Atrial Selectivity in Guinea Pig Hearts.

Background and Purpose: Prolongation of cardiac action potentials is considered antiarrhythmic in the atria but can be proarrhythmic in ventricles if the current carried by Kv11.1-channels (IKr) is inhibited. The current mediated by KCa2-channels, IKCa, is considered a promising new target for treatment of atrial fibrillation (AF). Selective inhibitors of IKr (dofetilide) and IKCa (AP14145) were used to compare the effects on ventricular and atrial repolarization. Ondansetron, which has been reported to be a potent blocker of both IKr and IKCa, was included to examine its potential atrial antiarrhythmic properties. Experimental Approach: The expression of KCa2- and Kv11.1-channels in the guinea pig heart was investigated using quantitative polymerase chain reaction (qPCR). Whole-cell patch clamp technique was used to investigate the effects of dofetilide, AP14145, and ondansetron on IKCa and/or IKr. The effect of dofetilide, AP14145, and ondansetron on atrial and ventricular repolarization was investigated in isolated hearts. A novel atrial paced in vivo guinea pig model was further validated using AP14145 and dofetilide. Key Results: AP14145 increased the atrial effective refractory period (AERP) without prolonging the QT interval with Bazett’s correction for heart rate (QTcB) both ex vivo and in vivo. In contrast, dofetilide increased QTcB and, to a lesser extent, AERP in isolated hearts and prolonged QTcB with no effects on AERP in the in vivo guinea pig model. Ondansetron did not inhibit IKCa, but did inhibit IKr in vitro. Ondansetron prolonged ventricular, but not atrial repolarization ex vivo. Conclusion and Implications: IKCa inhibition by AP14145 selectively increases atrial repolarization, whereas IKr inhibition by dofetilide and ondansetron increases ventricular repolarization to a larger extent than atrial repolarization.

Treatment of CaV1.2 Channelopathies May Be Complicated by Altered Channel Inactivation

Timothy Syndrome (TS) is a multisystem disorder, featuring neurological impairment, autism, and cardiac action-potential prolongation (long QT) with life-threatening arrhythmias. The underlying cause of TS was first described as a single point mutation (G406R) in CaV1.2 channels, which resulted in a disruption of both calcium- and voltage- dependent inactivation of the channel. Since this first discovery, a growing number of potentially arrhythmogenic CaV1.2 mutations have been found, increasing the need for targeted therapies. As many of these mutations have been shown to impair channel inactivation, calcium channel blockers (CCBs) such as verapamil and dihydropyridines (DHPs) represent a promising option for treatment, however, these CCBs often prove only partially effective for managing symptoms in these patients. We propose that this lack of efficacy is caused by attenuated channel inactivation in the mutant channels. In particular, verapamil and DHPs are known to preferentially block open and inactivated channels such that loss of entry into the inactivated state would decrease the efficacy of these CCBs on the mutant channels. Here, we demonstrate that mutations in CaV1.2 which diminish channel inactivation also decrease the extent of channel block by CCBs. Furthermore, we show that amount of channel block by these CCBs is strongly correlated to the fraction of inactivation in the channel, whereby efficacy of block is lowest in mutant channels with the lowest fraction of inactivation. This decreased efficacy of CCBs on mutant channels represents a major impediment to the treatment of TS and cardiac arrhythmias caused by mutations in CaV1.2. These results emphasize the need to tailor patient therapies to the specific channel deficit produced by each mutation.

Suppression of hERG K+ current and cardiac action potential prolongation by 4-hydroxynonenal via dual mechanisms

Oxidative stress under pathological conditions, such as ischemia/reperfusion and inflammation, results in the production of various reactive chemicals. Of these chemicals, 4-hydroxynonenal (4-HNE), a peroxidation product of ω6-polyunsaturated fatty acid, has garnered significant attention. However, the effect of 4-HNE on cardiac electrophysiology has not yet been reported. In the present study, we investigated the effects of 4-HNE on several cardiac ion channels, including human ether-a-go-go-related (hERG) channels, using the whole-cell patch clamp technique. Short-term exposure to 100 μM 4-HNE (4-HNE100S), which mimics local levels under oxidative stress, decreased the amplitudes of rapidly activating delayed rectifier K+ current (IKr) in guinea pig ventricular myocytes (GPVMs) and HEK293T cells overexpressing hERG (IhERG). MS analysis revealed the formation of 4-HNE-hERG adduct on specific amino acid residues, including C276, K595, H70, and H687. Long-term treatment (1–3 h) with 10 μM 4-HNE (4-HNE10L), suppressed IKr and IhERG, but not IKs and ICa,L. Action potential duration (APD) of GPVMs was prolonged by 37% and 64% by 4-HNE100S and 4-HNE10L, respectively. Western blot analysis using surface biotinylation revealed a reduction in mature membrane hERG protein after treatment with 4-HNE10L. Proteasomal degradation inhibitors, such as bortezomib, prevented the 4-HNE10L-induced decrease in mature hERG, suggesting a retrograde degradation of membrane hERG due to 4-HNE. Taken together, 4-HNE100S and 4-HNE10L suppressed IhERG via functional inhibition and downregulation of membrane expression of hERG, respectively. The exposure of 4-HNE under pathological oxidative stress may increase the risk of proarrhythmic events via APD prolongation.

Arrhythmogenesis in Timothy Syndrome is associated with defects in Ca2+-dependent inactivation

Timothy Syndrome (TS) is a multisystem disorder, prominently featuring cardiac action potential prolongation with paroxysms of life-threatening arrhythmias. The underlying defect is a single de novo missense mutation in CaV1.2 channels, either G406R or G402S. Notably, these mutations are often viewed as equivalent, as they produce comparable defects in voltage-dependent inactivation and cause similar manifestations in patients. Yet, their effects on calcium-dependent inactivation (CDI) have remained uncertain. Here, we find a significant defect in CDI in TS channels, and uncover a remarkable divergence in the underlying mechanism for G406R versus G402S variants. Moreover, expression of these TS channels in cultured adult guinea pig myocytes, combined with a quantitative ventricular myocyte model, reveals a threshold behaviour in the induction of arrhythmias due to TS channel expression, suggesting an important therapeutic principle: a small shift in the complement of mutant versus wild-type channels may confer significant clinical improvement.

In silico assessment of kinetics and state dependent binding properties of drugs causing acquired LQTS

The Kv11.1 or hERG potassium channel is responsible for one of the major repolarising currents (IKr) in cardiac myocytes. Drug binding to hERG can result in reduction in IKr, action potential prolongation, acquired long QT syndrome and fatal cardiac arrhythmias. The current guidelines for pre-clinical assessment of drugs in development is based on the measurement of the drug concentration that causes 50% current block, i.e., IC50. However, drugs with the same apparent IC50 may have very different kinetics of binding and unbinding, as well as different affinities for the open and inactivated states of Kv11.1. Therefore, IC50 measurements may not reflect the true risk of drug induced arrhythmias. Here we have used an in silico approach to test the hypothesis that drug binding kinetics and differences in state-dependent affinity will influence the extent of cardiac action potential prolongation independent of apparent IC50 values. We found, in general that drugs with faster overall kinetics and drugs with higher affinity for the open state relative to the inactivated state cause more action potential prolongation. These characteristics of drug-hERG interaction are likely to be more arrhythmogenic but cannot be predicted by IC50 measurement alone. Our results suggest that the pre-clinical assessment of Kv11.1-drug interactions should include descriptions of the kinetics and state dependence of drug binding. Further, incorporation of this information into sophisticated in silico models should be able to better predict arrhythmia risk and therefore more accurately assess safety of new drugs in development.

Human stem cell-derived cardiomyocytes detect drug-mediated changes in action potentials and ion currents

IntroductionIt has been proposed that proarrhythmia assessment for safety pharmacology testing includes the use of human pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) to detect drug-induced changes in cardiac electrophysiology. This study measured the actions of diverse agents on action potentials (AP) and ion currents recorded from hiPSC-CM. MethodsDuring AP experiments, the hiPSC-CM were paced at 1Hz during a baseline period, and when increasing concentrations of test compound were administered at 4-minute intervals. AP parameters, including duration (APD60 and APD90), resting membrane potential, rate of rise, and amplitude, were measured throughout the entire experiment. Voltage clamp experiments with E-4031 and nifedipine were similarly conducted. ResultsE-4031 produced a dose-dependent prolongation of cardiac action potential and blocked the hERG/IKr current with an IC50 of 17nM. At 3nM, dofetilide significantly increased APD90. Astemizole significantly increased APD60 and APD90 at 30nM. Terfenadine significantly increased APD90 at concentrations greater than 10nM. Fexofenadine, a metabolite of terfenadine, did not produce any electrophysiologic changes in cardiac action potentials. Flecainide produced a dose-dependent prolongation of the cardiac action potential at 1 and 3μM. Acute exposure to nifedipine significantly decreased APD60 and APD90 and produced a dose-dependent block of calcium current with an IC50 of 0.039μM. Verapamil first shortened APD60 and APD90 in a dose-dependent manner, until a compensating increase in APD90, presumably via hERG blockade, was observed at 1 and 3μM. Following a chronic exposure (20–24h) to clinically relevant levels of pentamidine, a significant increase in action potential duration was accompanied by early afterdepolarizations (EADs). DiscussionThese experiments show the ability of AP measured from hiPSC-CM to record the interactions of various ion channels via AP recording and avoid the limitations of using several single ion channel assays in a noncardiac tissue.

Moderate intensity physical activity prevents increased blood glucose concentrations, fat pad deposition and cardiac action potential prolongation following diet-induced obesity in a juvenile-adolescent rat model.

BackgroundBoth obesity and a lack of physical activity have been associated with an elevated risk of cardiovascular disease (CVD). The incidence of obesity is increasing, especially in juvenile-adolescents. While there is limited research examining the chronic effects of obesity in adolescent humans and animal models of this condition, little is also known concerning how moderate physical activity might prevent or attenuate secondary cardiovascular complications induced by obesity during adolescence. We investigated the effects of diet-induced obesity (consisting of a high-fat, high-carbohydrate diet (HFHC)) on biometric indices, vascular and airway function, cardiovascular function, systemic oxidative stress and markers of inflammation in a juvenile-adolescent rodent model. Four groups were used: control (CON), physical activity (PA) treated, HFHC and HFHC + PA (n = 16 per group). HFHC feeding started at 4 weeks of age for a period of 12 weeks. Physical activity treatment was initiated (PA and HFHC + PA groups) when the animals were 8 weeks of age, for 8 weeks.ResultsPhysical activity in juvenile-adolescent healthy rats showed no change in comparison to the CON group in all experimental parameters except for increases in lipid peroxidation, decreases in inflammatory cytokines, improvements in vascular reactivity and decreased atrial responses to positive chronotropic agents. The HFHC animals were mildly hyperglycemic, hypertensive, displayed renal hypertrophy and showed increased retroperitoneal fat pad deposition compared to the CON group. HFHC + PA rats were also hypertensive, however showed improvements in cardiac electrophysiology, body weight, fat pad deposition and inflammatory signaling, in comparison to the HFHC fed rats and CON animals.ConclusionIn conclusion, in a juvenile-adolescent animal model of diet-induced obesity engagement in physical activity is beneficial in reducing the inflammatory effects of obesity.

Abstract 157: Toll like Receptor 4 Activation Promotes Cardiac Arrhythmias By Decreasing The Transient Outward Potassium Current (ito) Through An Irf3 dependent And Myd88 independent Pathway

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll like receptors (TLR’s) play an important role in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias. Also the signaling pathway involved in these phenomena was studied. Action potentials, the presence of cardiac arrhythmias and transient outward K + current (I to ) were recorded in Wistar rat’s hearts after 24 h exposure to the TLR4 agonist ultrapure Lipopolysaccharide (LPS - 1μg/ml). TLR4 stimulation in vitro promotes a cardiac electrical remodeling that leads to cardiac action potential prolongation which evokes arrhythmic events such as delayed after depolarization (DAD's) and triggered activity. The perfusion of LPS (1μg/ml) during 30 minutes did not modify I to . Conversely, after 24 h of LPS incubation I to was reduced, with no changes in the biophysical properties of the current. Major changes in Ca 2+ cycling were not observed in ventricular myocytes after 24 h exposure to LPS; however, extrasystolic activity was present in a considerable number of cells (25%). Neither the blockade of Interleulink-1 receptor-associated kinase 4 nor nuclear factor kappa B (NF-kB) prevented the LPS effect on I to . However, interferon regulatory factor 3 (IRF3) inhibition prevented the effect of TLR4 activation on I to . Activation of TLR4 induced extrasystolic activity, longer AP duration and evoked DAD's and triggered activity because of a reduction in I to . The mechanism involved is MyD88-independent and IRF3-dependent.

Improvement of QT analysis for Evaluating the Proarrhythmic Risk of Drug: The Importance of Spatial and Temporal Dispersion of Repolarization

Cardiac arrhythmias, in particular life-threatening Torsades de Pointes (TdP) are serious adverse effects associated with a number of pharmaceuticals belonging to different classes. It is therefore critical to have reliable biomarkers for assessing this risk during pre-clinical testing of new compounds. Prolongation of cardiac action potential and consequently of the QT interval of the ECG is generally considered as indicative of a risk of arrhythmia. Evaluation of drug effects on QT in preclinical studies is therefore requested by ICH (International Conference on Harmonization) guideline (S7B). However there is now growing evidence that the prolongation of mean QT interval is not an accurate indicator of the risk of arrhythmia and that other parameters of cardiac repolarization are more predictive. They include instability of action potential duration and increase in transmural heterogeneity of myocardial repolarization (spatial variability), which can be investigated in specific in vitro tests. We have conducted a number of experiments in dogs for evaluating the ECG correlates of both markers in studies testing the effects of isoproterenol, cisapride, astemizole and hypokaliemia, which are known to be associated with a proarrhythmic risk. Instability of action potential duration is associated with an increase in the beat-to-beat (temporal) variability of the QT interval that is evaluated by calculating the coefficient of variation of this parameter or by plotting QT from each beat versus QT of previous beat. Spatial variability of repolarization correlates with changes in the morphology of the T wave, in particular increase in the interval between the peak and the end of the T wave and notching of this wave. In these experiments, we have therefore established a simple method for in vivo assessment of spatial and temporal variability of cardiac repolarization, which may help in the evaluation the pro-arrhythmic risk of drugs.

Stem Cell–Derived Cardiomyocytes as a Tool for Studying Proarrhythmia

Proarrhythmia is a leading cause of hospitalization for adverse drug events, and screening for proarrhythmic potential resulting from QT interval prolongation has become a major component of the development process for any new drug candidate. Conventional wisdom, now embedded in regulatory guidelines from the Food and Drug Administration and other regulatory agencies, holds that drug block of the rapid component of the cardiac delayed-rectifier potassium current, I Kr, is the major proximate mechanism underlying prolongation of cardiac action potentials, an effect that leads to early afterdepolarizations in vitro and is manifest on the surface ECG as QT prolongation and occasionally torsades de pointes.1,2 As a result, regulatory guidance suggests an early look at I Kr blocking potency of new drug entities, even recognizing that other mechanisms, some newly described,3,4 may contribute. Article see p 1677 However, the correlation between I Kr block and QT interval prolongation is imperfect. Multiple mechanisms may underlie this “disconnect,” and a reasonable generalization is that I Kr is but one (albeit important) component of normal cardiac repolarization, and abnormalities in expression or function of other components of repolarization may, in turn, modulate the net effect of I Kr block on action potentials in cardiac myocytes and on QT interval and arrhythmia susceptibility in the whole heart. The logical extension of this line of reasoning is that screening for proarrhythmic potential should include the evaluation of not only drug effects on individual ionic currents such as I Kr contributing to repolarization but also the integrated effect of drugs on action potentials in vitro and on the QT interval in animal models and in humans. In conventional drug development programs, this is commonly accomplished by comparing the effects of a candidate drug molecule on QT intervals in human subjects with …

Regulation of the Cardiac Sodium/Bicarbonate Cotransporter by Angiotensin II: Potential Contribution to Structural, Ionic and Electrophysiological Myocardial Remodelling

The sodium/ bicarbonate cotransporter (NBC) is, with the Na+/H+ exchanger (NHE), an important alkalinizing mechanism that maintains cellular intracellular pH (pHi). In the heart exists at least three isoforms of NBC, one that promotes the co-influx of 1 molecule of Na+ per 1molecule of HCO3-(electroneutral isoform; nNBC) and two others that generates the co-influx of 1 molecule of Na+ per 2 molecules of HCO3- (electrogenic isoforms; eNBC). In addition, the eNBC generates an anionic repolarizing current that modulate the cardiac action potential (CAP), adding to such isoforms the relevance to modulate the electrophysiological function of the heart. Angiotensin II (Ang II) is one of the main hormones that regulate cardiac physiology. The alkalinizing mechanisms (NHE and NBC) are stimulated by Ang II, increasing pHi and intracellular Na+ concentration, which indirectly, due to the stimulation of the Na+/Ca2+ exchanger (NCX) operating in the reverse form, leads to an increase in the intracellular Ca2+ concentration. Interestingly, it has been shown that Ang II exhibits an opposite effect on NBC isoforms: it activates the nNBC and inhibits the eNBC. This inhibition generates a CAP prolongation, which could directly increase the intracellular Ca2+ concentration. The regulation of the intracellular Na+ and Ca2+ concentrations is crucial for the cardiac cellular physiology, but these ions are also involved in the development of cardiac hypertrophy and the damage produced by ischemia-reperfusion, suggesting a potential role of NBC in cardiac diseases.

Identification and Characterization of a Compound That Protects Cardiac Tissue from Human Ether-à-go-go-related Gene (hERG)-related Drug-induced Arrhythmias

The human Ether-à-go-go-related gene (hERG)-encoded K(+) current, I(Kr) is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes I(Kr) block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC(70) of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC(50) of dofetilide from 38.7 to 76.3 nM. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.

Nonlinear Threshold Behavior in the Induction of Arrhythmias by Channels Bearing Timothy Syndrome Mutations

Timothy Syndrome (TS) is a multisystem disorder, featuring cardiac action-potential prolongation with paroxysms of life-threatening arrhythmias. The underlying defect is a single point mutation in CaV1.2 channels. Interestingly, recent clinical studies hint that overt disease manifestation might be mitigated by diminished expression of mutant channels. To gain insight, we undertook in-depth biophysical characterization of TS channels, and used this profile to refine a widely-utilized ventricular myocyte model (AJP 292:H2854). Simulations of variable levels of TS channels suggested a remarkable behavior. As expected, a modest increase in TS channels produced moderate action-potential prolongation (A1, control; A2, TS). By contrast, only a minute further increase in TS channels induced flagrant disturbances of excitability (A3), suggesting a nonlinear threshold for induction of frank arrhythmias. To explore this experimentally in cell networks, we variably expressed TS channels in monolayers of cultured guinea-pig ventricular myocytes. Indeed, conservative expression of TS channels yielded graded action-potential prolongation (B1, control; B2, TS), but small further elevation begat severe disturbances (B3). If like behavior exists in the heart, limited changes in TS expression may generate dramatic alterations in arrhythmogenic potential, an important principle for therapeutics.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Thermal adaptation of the crucian carp (Carassius carassius) cardiac delayed rectifier current, IKs, by homomeric assembly of Kv7.1 subunits without MinK

Ectothermic vertebrates experience acute and chronic temperature changes which affect cardiac excitability and may threaten electrical stability of the heart. Nevertheless, ectothermic hearts function over wide range of temperatures without cardiac arrhythmias, probably due to special molecular adaptations. We examine function and molecular basis of the slow delayed rectifier K(+) current (I(Ks)) in cardiac myocytes of a eurythermic fish (Carassius carassius L.). I(Ks) is an important repolarizing current that prevents excessive prolongation of cardiac action potential, but it is extremely slowly activating when expressed in typical molecular composition of the endothermic animals. Comparison of the I(Ks) of the crucian carp atrial myocytes with the currents produced by homomeric K(v)7.1 and heteromeric K(v)7.1/MinK channels in Chinese hamster ovary cells indicates that activation kinetics and pharmacological properties of the I(Ks) are similar to those of the homomeric K(v)7.1 channels. Consistently with electrophysiological properties and homomeric K(v)7.1 channel composition, atrial transcript expression of the MinK subunit is only 1.6-1.9% of the expression level of the K(v)7.1 subunit. Since activation kinetics of the homomeric K(v)7.1 channels is much faster than activation of the heteromeric K(v)7.1/MinK channels, the homomeric K(v)7.1 composition of the crucian carp cardiac I(Ks) is thermally adaptive: the slow delayed rectifier channels can open despite low body temperatures and curtail the duration of cardiac action potential in ectothermic crucian carp. We suggest that the homomeric K(v)7.1 channel assembly is an evolutionary thermal adaptation of ectothermic hearts and the heteromeric K(v)7.1/MinK channels evolved later to adapt I(Ks) to high body temperature of endotherms.

Pharmacological Modulations of Cardiac Ultra-Rapid and Slowly Activating Delayed Rectifier Currents: Potential Antiarrhythmic Approaches

Despite the emerging new insights into our understandings of the cellular mechanisms underlying cardiac arrhythmia, medical therapy for this disease remains unsatisfactory. Atrial fibrillation (AF), the most prevalent arrhythmia, is responsible for significant morbidity and mortality. On the other hand, ventricular fibrillation results in sudden cardiac deaths in many instances. Prolongation of cardiac action potential (AP) is a proven principle of antiarrhythmic therapy. Class III antiarrhythmic agents prolong AP and QT interval by blocking rapidly activating delayed rectifier current (I(Kr)). However, I(Kr) blocking drugs carry the risk of life-threatening proarrhythmia. Recently, modulation of atrial-selective ultra-rapid delayed rectifier current (I(Kur)), has emerged as a novel therapeutic approach to treat AF. A number of I(Kur) blockers are being evaluated for the treatment of AF. The inhibition of slowly activating delayed rectifier current (I(Ks)) has also been proposed as an effective and safer antiarrhythmic approach because of its distinguishing characteristics that differ in remarkable ways from other selective class III agents. Selective I(Ks) block may prolong AP duration (APD) at rapid rates without leading to proarrhythmia. This article reviews the pathophysiological roles of I(Kur) and I(Ks) in cardiac repolarization and the implications of newly developed I(Kur) and I(Ks) blocking agents as promising antiarrhythmic approaches. Several recent patents pertinent to antiarrhythmic drug development have been discussed. Further research will be required to evaluate the efficacy and safety of these agents in the clinical setting.

Mechanisms of ventricular arrhythmogenesis in mice following targeted disruption of KCNE1 modelling long QT syndrome 5

Mutations within KCNE1 encoding a transmembrane protein which coassembles with K+ channels mediating slow K+, I(Ks), currents are implicated in cardiac action potential prolongation and ventricular arrhythmogenicity in long QT syndrome 5. We demonstrate the following potentially arrhythmogenic features in simultaneously recorded, left ventricular, endocardial and epicardial monophasic action potentials from Langendorff-perfused murine KCNE1-/- hearts for the first time. (1) Prolonged epicardial (57.1 +/- 0.5 ms cf. 36.1 +/- 0.07 ms in wild-type (WT), P < 0.001; n = 5) and endocardial action potential duration at 90% repolarication (APD90) (54.4 +/- 2.4 ms cf. 48.5 +/- 0.3 ms, P < 0.05; n = 5). (2) Negative transmural repolarization gradients (DeltaAPD90: endocardial minus epicardial APD90) (-2.5 +/- 2.4 ms, compared with 12.4 +/- 1.1 ms in WT, P < 0.001; n = 5). (3) Frequent epicardial early afterdepolarizations (EADs) and spontaneous ventricular tachycardia (VT) in 4 out of 5 KCNE1-/- hearts but not WT (n = 5). EADs were especially frequent following temporary cessations of ventricular pacing. (4) Monomorphic VT lasting 1.36 +/- 0.2 s in 5 out of 5 KCNE1-/- hearts, following premature stimuli but not WT (n = 5). (5) Epicardial APD alternans. Perfusion of KCNE1-/- hearts with 1 mum nifedipine induced potentially anti-arrhythmic changes including: (1) restored epicardial APD90 (from 57.1 +/- 0.5 ms to 42.3 +/- 0.4 ms, P < 0.001; n = 5); (2) altered DeltaAPD90 to values (11.2 +/- 2.6) close to WT (P > 0.05; n = 5); (3) EAD suppression during both spontaneous activity and following cessation of ventricular pacing (n = 5) to give similar features to WT controls (n = 5); (4) suppression of programmed electrical stimulation-induced VT; and (5) suppression of APD alternans. These findings suggest arrhythmic effects of reduced outward currents expected in KCNE1-/- hearts and their abolition by antagonism of inward L-type Ca2+ current.

Inhibitory Effect of Thiopental on Ultra-rapid Delayed Rectifier K+ Current in H9c2 Cells

Using the whole-cell voltage clamp technique, we investigated the effects of thiopental on membrane currents in H9c2 cells, a cell line derived from embryonic rat heart. Thiopental blocked a rapidly activating, very slowly-inactivating ultra-rapid type IKur-like outward K+ current in a concentration-dependent manner. The half-maximal concentration (IC50) of thiopental was 97 µM with a Hill coefficient of 1.2. The thiopental-sensitive current was also blocked by high concentrations of nifedipine (IC50 = 9.1 µM) and 100 µM chromanol 293B, a blocker of slowly activating delayed rectifier K+ current (IKs), but was insensitive to E-4031, an inhibitor of rapidly activating delayed rectifier K+ current (IKr). TEA (tetraethylammonium) at 5 mM and 4-AP (4-aminopiridine) at 1 mM reduced the K+ current to 30.8 ± 12.2% and 20.5 ± 6.5% of the control, respectively. Using RT-PCR, we detected mRNAs of Kv2.1, Kv3.4, Kv4.1, and Kv4.3 in H9c2 cells. Among those, Kv2.1 and Kv3.4 have IKur-type kinetics and are therefore candidates for thiopental-sensitive K+ channels in H9c2 cells. This is the first report showing that thiopental inhibits IKur. This effect of thiopental may be involved in its reported prolongation of cardiac action potentials.