Ultra-rapid Delayed Rectifier Potassium Current Research Articles

Сardioprotective agents with biaromatic structure. Part 5. Potassium Kv1.5-channels blockers

The Kv1.5 potassium channel provides an ultra-rapid delayed rectifier potassium current, IKur, that acts selectively in human atrial cells. This makes selective Kv1.5 blockade a promising approach to control atrial arrhythmias without the adverse ventricular effects associated with classical hERG-subtype potassium channel blockers (Kv11.1). This review considers all currently known Kv1.5-channel blockers with a biaromatic structure and data on their biological properties. For many of the Kv1.5-selective compounds studied, the ability to prevent the development of atrial arrhythmias without affecting ventricular refractoriness was confirmed.

The Effect of the Tongyang Huoxue Recipe (TYHX) on the I to/I Kur in Ischemia/Reperfusion Sinoatrial Node Cells.

The transient outward potassium current (I to) and the ultrarapid delayed rectifier potassium current (I Kur) are major potassium currents involved in the repolarization process of sinoatrial node cells (SNCs). The SNCs of neonatal rats were divided into control, ischemia/reperfusion (I/R), I/R+blank serum, and Tongyang Huoxue recipe (TYHX) serum groups. I to and I Kur were recorded using the whole cell patch-clamp technique, and the current-voltage (I-V), steady-state activation (SSA), steady-state inactivation (SSI), and recovery from inactivation (RFI) curves were plotted, respectively. Compared to the control group, both the peak current density and the current density at the voltage of I to and I Kur decreased obviously in SNCs after simulated I/R, the SSA curves moved right, and the SSI curves moved left. After TYHX was added to the extracellular solution of SNCs, both the peak current density and the current density at the voltage of I to and I Kur increased significantly, the SSA curves moved left, and the SSI curves moved right with a significant difference of V 1/2. The recovery from the I Kur RFI curves was slightly restored, and the I to curves did not change. TYHX increases the peak current density, accelerates the activation, and decreases the inactivation of the I to and I Kur. This may be the mechanism of TYHX in shortening the action potential duration of repolarization, which accelerates spontaneous pulsation.

Open channel block of Kv1.5 channels by HMQ1611.

Kv1.5 channels conduct the ultra-rapid delayed rectifier potassium current (I Kur). Pharmacological blockade of human Kv1.5 (hKv1.5) has been regarded as an effective treatment of re-entrant based atrial fibrillation, because Kv1.5 is highly expressed in human cardiac atria but scarcely in ventricles. The Kv1.5 blockade is also expected to be used in cancer therapeutics since Kv1.5 is overexpressed in some types of human tumors. Here, we investigated the blockade of hKv1.5 channels by HMQ1611, a symmetrical biphenyl derivative. hKv1.5 channels were heterologously expressed in Chinese hamster ovary cells. The effects of HMQ1611 on wild-type and 13 hKv1.5 mutant channels were examined using the whole-cell patch-clamp method, and molecular docking simulation was conducted to predict the docking position of HMQ1611 within Kv1.5 channels. We showed that HMQ1611 reversibly inhibited the hKv1.5 current in a concentration-dependent manner (IC50 = 2.07 μM). HMQ1611 blockade of hKv1.5 current developed with time during depolarizing voltage-clamp steps, and this blockade was also voltage-dependent with a steep increase over the voltage range for channel openings. HMQ1611 inhibition was significantly reduced in the T479A, T480A, V505A, I508A, L510A, V512A, and V516A hKv1.5 mutant channels. Molecular docking analysis predicted that V505, V512, and T480 were involved in the blocking action of HMQ1611 on hKv1.5 channels. These results suggest that HMQ1611 inhibits hKv1.5 currents as an open channel blocker. Amino acid residues located at the base of the selectivity filter (T479 and T480) and in the S6 segment (V505, I508, L510, V512, and V516) of hKv1.5 appear to constitute potential binding sites for HMQ1611.

Treating atrial fibrillation with radiofrequency ablation to reverse changes in microRNAs regulating the ion-channel proteins.

To investigate the possible molecular mechanisms of radiofrequency ablation (RFA) for treating atrial fibrillation (AF) and the microRNA (miRNA) target for intervention in the future. We examined the changes in miRNAs regulating the atrial ion-channel proteins across the whole genome. We compared findings from 90 AF patients with those from 90 healthy subjects before RFA and three months after RFA. Twenty-one miRNAs regulating ion-channel proteins were differentially expressed more than ten-fold, and the findings were completely reversed after RFA as compared with the pre-RFA results. The colonial regulating effects of miRNAs regulating the outward K+ current channels such as those for the ultra-rapid delayed rectifier potassium current (Ikur), voltage-dependent delayed rectifier potassium current (Ikr), and delayed rectifier potassium channel current (Iks) were more unanimous and stronger, while this was not the case for miRNAs regulating the L-type Ca2+ current and INa current channels. Generally, miR-1266 levels were increased in the blood but down-regulated in the rheumatic atrial tissue, while a dual luciferase test indicated that SCN5A was the direct target gene of miR-1266. Using RFA to treat AF may have an impact via reversing the changes in miRNAs regulating the ion-channel proteins, especially for outward K+ current channels such as Ikur, Ikr, and Iks, which may play a major role in electrical remodeling in AF. It may be that miR-1266 is an antiarrhythmic miRNA and an AF intervention target in the future (Tab. 2, Fig. 4, Ref. 46).

Tyrosine-protein kinase Src regulates Kv1.5 channel activity and membrane expression through interaction with the N-terminus of the channel

Kv1.5 is a voltage-gated potassium channel that generates the ultra-rapid delayed rectifier potassium current (IKur) important in the repolarization of the atrial action potential. Malfunction of the Kv1.5 channel often results in atrial fibrillation (AFib). A reduction in Kv1.5 current (IKv1.5) occurs upon activation of the endogenous tyrosine-protein kinase Src. The Src SH3 domain binds to proline-rich motifs located within the N-terminus of Kv1.5. Disruption of these binding motifs has been involved in the development of familial AFib. The mechanism underlying the reduction of IKv1.5 upon Src activation has not yet been established and the relationship between Kv1.5 and Src is poorly understood. Therefore, the present study aims to further elucidate the mechanism behind IKv1.5 reduction. The hypothesis that Src regulates Kv1.5 activity by altering the density of mature membrane-localized channels was tested using whole-cell voltage clamp and Western blot analysis. We demonstrate that Src tonically inhibits Kv1.5 activity and decreases the density of mature membrane-localized channels. Kv1.5 channels possessing mutations within the Src binding motifs were also investigated and it was determined that each binding motif contributes to the Kv1.5-Src relationship, however, the binding of Src to an individual motif is sufficiently effective. Our findings indicate that Src regulates Kv1.5 through an interaction with the N-terminal binding motifs and suggests that the inhibition of forward trafficking may be involved in the underlying mechanism. (Supported by the Heart and Stroke foundation of Canada and The Canadian Institutes of Health Research).

Availability of a novel cardiotoxicity evaluation system using human induced pluripotent stem cell-derived atrial-like myocytes

It is reported that the incidence of atrial arrhythmias has been increasing year by year and it might increase from now on. Although not only aging but pharmaceutical drug treatments might relate to atrial arrhythmias, experimental method to detect drug-induced atrial arrhythmias has not been established so far. Therefore, we induced differentiation of atrial-like cardiomyocytes from human induced pluripotent stem (iPS) cell, and clarified their characteristics and verified their dug responsiveness. Atrial-like cardiomyocytes were induced by adding retinoic acid (RA) during the process of myocardial differentiation, and their character was compared to RA-untreated cardiomyocytes. In gene expression and membrane potential analysis, it was confirmed that the cells with or without RA treatment have the characters of atrial or ventricular like cardiomyocytes, respectively. In addition, it was also confirmed that atrial-like cardiomyocytes induced reentry-like conduction disorder, which is atrial arrhythmias. Furthermore, as a result of examining the responsiveness of various ion channel inhibitors using these cells, the inhibition of ultra-rapid delayed rectifier potassium current (IKur) specifically existed in atrial muscle induced prolongation of PWD30cF (membrane potential duration at 30% depolarization corrected by Fridericia formula) only in atrial-like cardiomyocytes. In addition, ventricular-like cardiomyocytes alone exhibited an early after depolarization by treatment of rapid rectifier potassium current (IKr) inhibitor which induced ventricular arrhythmia in clinical situation. Based on above evidences, current evaluation systems using human iPS cell-derived atrial-like cardiomyocytes might be a valuable tool for drug-induced atrial arrhythmias.

Macrophage migration inhibitory factor increases atrial arrhythmogenesis through CD74 signaling

Macrophage migration inhibitory factor (MIF), a pleiotropic inflammatory cytokine, is highly expressed in patients with atrial fibrillation (AF). CD74 (major histocompatibility complex, class II invariant chain) is the main receptor for MIF. However, the role of the MIF/CD74 axis in atrial arrhythmogenesis is unclear. In this study, we investigated the effects of MIF/CD74 signaling on atrial electrophysiological characteristics and determined its underlying mechanisms. Confocal fluorescence microscopy, patch clamp, and western blot analysis were used to study calcium homeostasis, ionic currents, and calcium-related signaling in MIF-treated HL-1 atrial cardiomyocytes with or without anti-CD74 neutralized antibodies treatment. Furthermore, electrocardiographic telemetry recording and echocardiography were obtained from mice treated with MIF. Compared with controls, MIF-treated HL-1 myocytes had increased calcium transients, sarcoplasmic reticulum (SR) calcium content, Na+/Ca2+ exchanger (NCX) efflux rate, calcium leak, transient outward potassium current, and ultra-rapid delayed rectifier potassium current. Furthermore, MIF could induce expression of SR Ca2+ATPase, NCX, phosphorylation of ryanodine receptor 2 (RyR2), and activation of calcium/calmodulin kinase II (CaMKII) when compared with control cells. MIF-mediated electrical dysregulation and CaMKII-RyR2 signaling activation were attenuated through blocking of CD74. Moreover, MIF-injected mice had lesser left atrium fractional shortening, greater atrial fibrosis, and atrial ectopic beats than control (nonspecific immunoglobulin treated) or MIF combined with anti-CD74 neutralized antibody-treated mice. Consequently, our study on MIF/CD74 signaling has pointed out a new potential therapeutic intervention of AF patients with MIF elevation.

Long-term 4-AP treatment facilitates functional expression of human Kv1.5 channel

The human Kv1.5 channel (hKv1.5) produces the ultrarapid delayed rectifier potassium current (IKur), which is important for determining the repolarization of action potential in the cardiac atrium. However, the expression of IKur is reduced in patients with chronic atrial fibrillation. 4-Aminopyridine (4-AP) can specifically suppress IKur, suggesting that it modifies hKv1.5 as a chaperone molecule. Herein, the effects of long-term 4-AP treatment on hKv1.5 protein expression and function were investigated in HEK cells. 4-AP treatment (24 h) improved hKv1.5 protein levels, promoted hKv1.5 glycosylation, and facilitated the hKv1.5 current in a time-dependent manner. Long-term 4-AP treatment also markedly enhanced hKv1.5 localization in the cell membrane, endoplasmic reticulum, and Golgi. Importantly, the Ile508 residue located in the hKv1.5 channel pore was found to be important for 4-AP inhibitory activity. These results provide insight into developing hKv1.5 channel blocker that can functionally rescue IKur in patients with chronic atrial fibrillation.

Epigallocatechin-3-gallate modulates arrhythmogenic activity and calcium homeostasis of left atrium

BackgroundAtrial fibrillation (AF) is the commonest sustained arrhythmia, and increases the risk of stroke, heart failure, and mortality. Calcium (Ca2+) overload and oxidative stress are thought to participate in the pathogenesis of AF. Epigallocatechin-3-gallate (EGCG) has an antioxidative effect and been shown to be beneficial in promoting cardiovascular health. However, it is not clear if EGCG directly modulates the electrophysiological characteristics and Ca2+ homeostasis of the left atrium (LA). Methods and resultsConventional microelectrodes, whole-cell patch-clamp, and Fluo-3 fluorometric ratio technique were performed using the isolated rabbit LA preparations or isolated single LA cardiomyocytes before and after EGCG treatment. EGCG (0.01, 0.1, 1, and 10μM) which concentration-dependently decreased the APD20 by 13±8%, 25±5%, 31±6%, and 37±5%, APD50 by 9±8%, 22±6%, 32±7%, and 40±4%, and APD90 by 2±12%, 9±8%, 24±10%, and 34±5% in LA preparations. EGCG (0.1μM) decreased the late sodium (Na+) current, L-type Ca2+ current, nickel-sensitive Na+-Ca2+ exchanger current, and transient outward current, but did not change the Na+ current and ultra-rapid delayed rectifier potassium current in LA cardiomyocytes. EGCG decreased intracellular Ca2+ transient and sarcoplasmic reticulum Ca2+ content in LA cardiomyocytes. Furthermore, EGCG decreased isoproterenol (ISO, 1μM)-induced burst firing. KT5823 (1μM) or KN93 (1μM) decreased the incidences of ISO-induced LA burst firing, which became lower with EGCG treatment. H89 (10μM) and KN92 (1μM) did not suppress the incidence of ISO-induced LA burst firing. However, EGCG decreased the incidences of ISO-induced LA burst firing in the presence of H89 or KN92. ConclusionEGCG directly regulates LA electrophysiological characteristics and Ca2+ homeostasis, and suppresses ISO-induced atrial arrhythmogenesis through inhibiting Ca2+/calmodulin or cGMP-dependent protein kinases.

Regulation of human cardiac Kv1.5 channels by extracellular acidification.

Human Kv1.5 channels (hKv1.5) conduct the ultra-rapid delayed rectifier potassium current (I Kur), which plays an important role in action potential repolarization of atrial myocytes. The present study was undertaken to examine the effects of acidic pH on hKv1.5 wild-type (WT) and its pore mutant channels heterologously expressed in Chinese hamster ovary (CHO) cells using site-directed mutagenesis combined with whole-cell patch-clamp technique. Both extracellular and intracellular acidifications equally and reversely reduced the amplitude of hKv1.5 currents. The extracellular acidification significantly shifted the voltage dependence of current activation to more depolarized potentials and accelerated deactivation kinetics of the current. The ancillary β subunits Kvβ1.3 and Kvβ1.2, known to modify the pharmacological sensitivities of hKv1.5, enhanced the extracellular proton-induced inhibitory effect on hKv1.5 current. In addition, several mutants (T462C, T479A, T480A, and I508A) exhibited significantly higher sensitivity to acidic pH-induced inhibition compared with WT channel, whereas the inhibitory effect of acidic pH was markedly reduced in H463G mutant. These observations indicate that (1) extracellular acidification modifies hKv1.5 gating and activity, (2) β subunits and several residues (T462, T479, T480, and I508) play critical roles in determining the sensitivity of the channel to acidic exposure, and (3) H463 may be a critical sensor for the channel inhibition by extracellular protons.

ZFHX3 knockdown increases arrhythmogenesis and dysregulates calcium homeostasis in HL-1 atrial myocytes

BackgroundZFHX3 plays an important role in the genesis of atrial fibrillation. However, the atrial electrophysiological effects of ZFHX3 are not clear. This study sought to investigate roles of ZFHX3 in atrial electrophysiology and calcium homeostasis by using HL-1 atrial myocytes knocked-down with ZFHX3. MethodsPatch clamp, confocal fluorescence microscopy and Western blot were used to study electrical activity, ionic currents, calcium homeostasis and protein expressions in stable ZFHX3 shRNA cells. ResultsAs compared to control, ZFHX3 shRNA cells with 28% decline of ZFHX3 protein had a larger sarcoplasmic reticulum Ca2+ content by 62%, Ca2+ transient by 20%, and calcium leak by 75%. ZFHX3 shRNA cells (n=35) had shorter action potential duration (APD) at 50% (14.7±0.9 versus 20.3±1.4ms, P<0.005), and 20% (6.1±0.3 versus 8.3±0.8ms, P<0.005) repolarization than control cells (n=30). ZFHX3 shRNA cells (n=10) had larger amplitudes of isoproterenol (1μM)-induced delayed afterdepolarization (14.1±0.9 versus 7.2±0.2mV, P<0.05) than control cells (n=10). Besides, acetylcholine (3μM) shortened APD at 90% repolarization to a greater extent (19±4% versus 7±2%, P<0.01) in ZFHX3 shRNA cells (n=11) than in control cells (n=12). In addition, ZFHX3 shRNA cells had increased expressions of SERCA2a, ryanodine receptor, Kv1.4, Kv1.5 and Kir3.4. Moreover, ZFHX3 shRNA cells had a larger SERCA2a activity, ultra-rapid delayed rectifier potassium currents, transient outward currents and acetylcholine-sensitive potassium currents. ConclusionsZFHX3 knock-down in atrial myocytes dysregulated calcium homeostasis and increased atrial arrhythmogenesis, which may contribute to the occurrence of AF.

Chronic obstructive sleep apnea causes atrial remodeling in canines: mechanisms and implications.

Obstructive sleep apnea (OSA) is closely related to atrial fibrillation (AF). However, the roles and mechanisms of chronic OSA in atrial remodeling are still unclear. Canine model of chronic OSA was simulated by stopping the ventilator and closing the airway for 4 h per day and lasting for 12 weeks. AF inducibility and duration was increased while atrial effective refractory period (AERP) was shortened after chronic apnea. Meanwhile, upregulation of proteins encoding inward rectifier K(+) current (IK1), delayed rectifier K(+) current (IKr and IKs), acetylcholine activated K(+) current (IKACh), transient outward K(+) current (Ito) and ultra-rapid delayed rectifier potassium current (IKur) as well as downregulation of protein encoding L-type Ca(2+) current (ICa,L) were found after chronic OSA. Besides abnormal electrical activity, chronic OSA induced apoptosis and interstitial fibrosis of atrial myocytes, which was partly mediated by caspase 9, phosphorylation of extracellular-regulated kinase 1/2, and α-smooth muscle actin. In addition, atrial sympathetic and parasympathetic hyperinnervation were found manifesting by enhanced growth-associated protein 43, tyrosine hydroxylase and elevated choline acetyltransferase. Moreover, protein expression of β1, β2, and M2 receptor were markedly increased by chronic OSA. In summary, we firstly demonstrated in canine model that chronic OSA could shorten AERP and lead to altered expression of important channel proteins, moreover, induce atrial structure remodeling by increased atrial apoptosis, fibrosis, and autonomic remodeling, eventually promoting the development of a substrate of AF. Our findings suggested that reversing atrial remodeling might be a potential therapeutic strategy for OSA-induced AF.

Rotor Termination Is Critically Dependent on Kinetic Properties of IKur Inhibitors in an In Silico Model of Chronic Atrial Fibrillation

Inhibition of the atrial ultra-rapid delayed rectifier potassium current (I Kur) represents a promising therapeutic strategy in the therapy of atrial fibrillation. However, experimental and clinical data on the antiarrhythmic efficacy remain controversial. We tested the hypothesis that antiarrhythmic effects of I Kur inhibitors are dependent on kinetic properties of channel blockade. A mathematical description of I Kur blockade was introduced into Courtemanche-Ramirez-Nattel models of normal and remodeled atrial electrophysiology. Effects of five model compounds with different kinetic properties were analyzed. Although a reduction of dominant frequencies could be observed in two dimensional tissue simulations for all compounds, a reduction of spiral wave activity could be only be detected in two cases. We found that an increase of the percent area of refractory tissue due to a prolongation of the wavelength seems to be particularly important. By automatic tracking of spiral tip movement we find that increased refractoriness resulted in rotor extinction caused by an increased spiral-tip meandering. We show that antiarrhythmic effects of I Kur inhibitors are dependent on kinetic properties of blockade. We find that an increase of the percent area of refractory tissue is the underlying mechanism for an increased spiral-tip meandering, resulting in the extinction of re-entrant circuits.

Leptin modulates electrophysiological characteristics and isoproterenol-induced arrhythmogenesis in atrial myocytes

BackgroundObesity is an important risk factor for atrial fibrillation (AF). Leptin is an important adipokine. However, it is not clear whether leptin directly modulates the electrophysiological characteristics of atrial myocytes.ResultsWhole cell patch clamp and indo-1 fluorescence were used to record the action potentials (APs) and ionic currents in isolated rabbit left atrial (LA) myocytes incubated with and without (control) leptin (100 nM) for 1 h to investigate the role of leptin on atrial electrophysiology. Leptin-treated LA myocytes (n = 19) had longer 20% of AP duration (28 ± 3 vs. 21 ± 2 ms, p < 0.05), but similar 50% of AP duration (51 ± 4 vs. 50 ± 3 ms, p > 0.05), and 90% of AP duration (89 ± 5 vs. 94 ± 4 ms, p > 0.05), as compared to the control (n = 22). In the presence of isoproterenol (10 nM), leptin-treated LA myocytes (n = 21) showed a lower incidence (19% vs. 54.2%, p < 0.05) of delayed afterdepolarization (DAD) than the control (n = 24). Leptin-treated LA myocytes showed a larger sodium current, but a smaller ultra-rapid delayed rectifier potassium current, and sodium-calcium exchanger current than the control. Leptin-treated and control LA myocytes exhibited a similar late sodium current, inward rectifier potassium current, transient outward current and L-type calcium current. In addition, the leptin-treated LA myocytes (n = 38) exhibited a smaller intracellular Ca2+ transient (0.21 ± 0.01 vs. 0.26 ± 0.01 R410/485, p < 0.05) and sarcoplasmic reticulum Ca2+ content (0.35 ± 0.02 vs. 0.43 ± 0.03 R410/485, p < 0.05) than the control LA myocytes (n = 42).ConclusionsLeptin regulates the LA electrophysiological characteristics and attenuates isoproterenol-induced arrhythmogenesis.

Rivaroxaban modulates electrical and mechanical characteristics of left atrium

BackgroundRivaroxaban reduces stroke in patients with atrial fibrillation (AF). Left atrium (LA) plays a critical role in the pathophysiology of AF. However, the electromechanical effects of rivaroxaban on LA are not clear.ResultsConventional microelectrodes and a whole-cell patch-clamp were used to record the action potentials (APs) and ionic currents in rabbit LA preparations and isolated single LA cardiomyocytes before and after the administration of rivaroxaban. Rivaroxaban (10, 30, 100, and 300 nM) concentration-dependently reduced LA (n = 7) AP durations at 90% repolarization (APD90) from 76 ± 2 to 79 ± 3, 67 ± 4 (P < 0.05, vs. control), 59 ± 5, (P < 0.01, vs. control), and 56 ± 4 ms (P < 0.005, vs. control), respectively. Rivaroxaban (10, 30, 100, and 300 nM) concentration-dependently increased the LA (n = 7) diastolic tension by 351 ± 69 (P < 0.05, vs. control), 563 ± 136 (P < 0.05, vs. control), 582 ± 119 (P < 0.05, vs. control), and 603 ± 108 mg (P < 0.005, vs. control), respectively, but did not change LA contractility. In the presence of L-NAME (100 μM) and indomethacin (10 μM), additional rivaroxaban (300 nM) treatment did not significantly further increase the LA (n = 7) diastolic tension, but shortened the APD90 from 73 ± 2 to 60 ± 6 ms (P < 0.05, vs. control). Rivaroxaban (100 nM) increased the L-type calcium current and ultra-rapid delayed rectifier potassium current, but did not change the transient outward potassium current in isolated LA cardiomyocytes.ConclusionsRivaroxaban modulates LA electrical and mechanical characteristics with direct ionic current effects.

Acacetin causes a frequency- and use-dependent blockade of hKv1.5 channels by binding to the S6 domain

We have demonstrated that the natural flavone acacetin selectively inhibits ultra-rapid delayed rectifier potassium current (IKur) in human atria. However, molecular determinants of this ion channel blocker are unknown. The present study was designed to investigate the molecular determinants underlying the ability of acacetin to block hKv1.5 channels (coding IKur) in human atrial myocytes using the whole-cell patch voltage-clamp technique to record membrane current in HEK 293 cells stably expressing the hKv1.5 gene or transiently expressing mutant hKv1.5 genes generated by site-directed mutagenesis. It was found that acacetin blocked hKv1.5 channels by binding to both closed and open channels. The blockade of hKv1.5 channels by acacetin was use- and frequency-dependent, and the IC50 of acacetin for inhibiting hKv1.5 was 3.5, 3.1, 2.9, 2.1, and 1.7μM, respectively, at 0.2, 0.5, 1, 3, and 4Hz. The mutagenesis study showed that the hKv1.5 mutants V505A, I508A, and V512A in the S6-segment remarkably reduced the channel blocking properties by acacetin (IC50, 29.5μM for V505A, 19.1μM for I508A, and 6.9μM for V512A). These results demonstrate the novel information that acacetin mainly blocks open hKv1.5 channels by binding to their S6 domain. The use- and rate-dependent blocking of hKv1.5 by acacetin is beneficial for anti-atrial fibrillation.

Novel KCNA5 mutation implicates tyrosine kinase signaling in human atrial fibrillation

Emerging evidence has strongly implicated hereditary determinants for atrial fibrillation (AF). Loss-of-function mutations in KCNA5 encoding the ultrarapid delayed rectifier potassium current I(Kur) have been identified in AF families. The purpose of this study was to determine the clinical and biophysical phenotypes in a KCNA5 mutation with deletion of 11 amino acids in the N-terminus of the protein, which was identified in patients with lone AF. Patients with AF confirmed by ECG were prospectively enrolled in the Vanderbilt AF Registry, which comprises clinical and genetic databases. A KCNA5 mutation was generated by mutagenesis for electrophysiologic characterization. We identified a novel 33-bp coding region deletion in two Caucasian probands. One proband was part of a kindred that included four other members with AF, and all were mutation carriers. The mutation results in deletion of 11 amino acids in the N-terminus of the protein, a proline-rich region as a binding site for Src homology 3 (SH3) domains associated with Src-family protein tyrosine kinase (TK) pathway. In transfected cells, the mutant caused approximately 60% decreased I(Kur) versus wild-type (WT) (75 +/- 8 pA/pF vs 180 +/- 15 pA/pF, P <.01) and dominant-negative effect on WT current (105 +/- 10 pA/pF, P <.01). Pretreatment with the Src inhibitor PP2 prevented v-Src TK from 90% suppressed WT current. In contrast, the mutant channel displayed no response to v-Src TK. Our data implicate abnormal atrial repolarization control due to variable TK signaling as a mechanism in familial AF and thereby suggest a role for modulation of this pathway in AF and its treatment.

Atrial-selective sodium channel block as a novel strategy for the management of atrial fibrillation

Pharmacological management of atrial fibrillation (AF) remains an important unmet medical need. Because available drugs for rhythm control of AF are often associated with a significant risk for development of ventricular arrhythmias or extracardiac toxicity, recent drug development has focused on agents that are atrial selective. Inhibition of the ultrarapid delayed rectifier potassium current ( I Kur), a current exclusive to atria, is an example of an atrial-selective approach. Recent studies, however, have shown that loss-of-function mutations in KCNA5, the gene that encodes K V1.5, the α subunit of the I Kur channel, is associated with the development of AF and that inhibition of I Kur can promote the induction of AF in experimental models. Another potential atrial-selective approach has recently been identified. Experimental studies have demonstrated important atrioventricular differences in the biophysical properties of the sodium channel and have identified sodium channel blockers that can exploit electrophysiological distinctions between atria and ventricles. Atrial-selective/predominant sodium channel blockers such as ranolazine effectively suppress AF in experimental models involving canine-isolated right atrial preparations at concentrations that produce little to no effect on electrophysiological parameters in ventricular myocardium. Chronic administration of amiodarone was also found to exert atrial-selective depression of I Na-dependent parameters and thus to prevent the induction of AF. Ranolazine and amiodarone have in common the ability to rapidly dissociate from the sodium channel and to prolong the atrial action potential duration via inhibition of I Kr. Our observations suggest that atrial-selective sodium channel block may be a fruitful strategy for the management of AF.

Possible role of the ultra-rapid delayed rectifier potassium current (IKur) in action potential repolarization in rabbit heart

Possible role of the ultra-rapid delayed rectifier potassium current (IKur) in action potential repolarization in rabbit heart

Inhibitory actions of the phosphatidylinositol 3‐kinase inhibitor LY294002 on the human Kv1.5 channel

Kv1.5 channels conduct the ultra-rapid delayed rectifier potassium current (I(Kur)), and in humans, Kv1.5 channels are highly expressed in cardiac atria but are scarce in ventricles. Pharmacological blockade of human Kv1.5 (hKv1.5) has been regarded as effective for prevention and treatment of re-entry-based atrial tachyarrhythmias. Here we examined blockade of hKv1.5 channels by LY294002, a well-known inhibitor of phosphatidylinositol 3-kinase (PI3K). hKv1.5 channels were heterologously expressed in Chinese hamster ovary cells. Effects of LY294002 on wild-type and mutant (T462C, H463C, T480A, R487V, A501V, I502A, I508A, L510A and V516A) hKv1.5 channels were examined by using the whole-cell patch-clamp method. LY294002 rapidly and reversibly inhibited hKv1.5 current in a concentration-dependent manner (IC(50) of 7.9 micromol.L(-1)). In contrast, wortmannin, a structurally distinct inhibitor of PI3K, had little inhibitory effect on hKv1.5 current. LY294002 block of hKv1.5 current developed with time during depolarizing voltage-clamp steps, and this blockade was also voltage-dependent with a steep increase over the voltage range for channel openings. The apparent binding (k(+1)) and unbinding (k(-1)) rate constants were calculated to be 1.6 micromol.L(-1) (-1).s(-1) and 5.7 s(-1) respectively. Inhibition by LY294002 was significantly reduced in several hKv1.5 mutant channels: T480A, R487V, I502A, I508A, L510A and V516A. LY294002 acts directly on hKv1.5 currents as an open channel blocker, independently of its effects on PI3K activity. Amino acid residues located in the pore region (Thr480, Arg487) and the S6 segment (Ile502, Ile508, Leu510, Val516) appear to constitute potential binding sites for LY294002.