Cardiac Kv1 Research Articles

Crosstalk between PKA and PIAS3 regulates cardiac Kv4 channel SUMOylation

Post-translational SUMOylation of nuclear and cytosolic proteins maintains homeostasis in eukaryotic cells and orchestrates programmed responses to changes in metabolic demand or extracellular stimuli. In excitable cells, SUMOylation tunes the biophysical properties and trafficking of ion channels. Ion channel SUMOylation status is determined by the opposing enzyme activities of SUMO ligases and deconjugases. Phosphorylation also plays a permissive role in SUMOylation. SUMO deconjugases have been identified for several ion channels, but their corresponding E3 ligases remain unknown. This study shows PIAS3, a.k.a. KChAP, is a bona fide SUMO E3 ligase for Kv4.2 and HCN2 channels in HEK cells, and endogenous Kv4.2 and Kv4.3 channels in cardiomyocytes. PIAS3-mediated SUMOylation at Kv4.2-K579 increases channel surface expression through a rab11a-dependent recycling mechanism. PKA phosphorylation at Kv4.2-S552 reduces the current mediated by Kv4 channels in HEK293 cells, cardiomyocytes, and neurons. This study shows PKA mediated phosphorylation blocks Kv4.2-K579 SUMOylation in HEK cells and cardiomyocytes. Together, these data identify PIAS3 as a key downstream mediator in signaling cascades that control ion channel surface expression.

Inhibition of Cardiac Kv4.3/KChIP2 Channels by Sulfonylurea Drug Gliquidone.

The Kv4.3 channel features fast N-type inactivation and also undergoes a slow C-type inactivation. The gain-of-function mutations of Kv4.3 channels cause an inherited disease called Brugada syndrome (BrS), characterized by a shortened duration of cardiac action potential repolarization and ventricular arrhythmia. The sulfonylurea drug gliquidone, an ATP-dependent K+ channel antagonist, is widely used for the treatment of type 2 diabetes. Here, we report a novel role of gliquidone in inhibiting Kv4.3 and Kv4.3/KChIP2 channels that encode the cardiac transient outward K+ currents responsible for the initial phase of action potential repolarization. Gliquidone results in concentration-dependent inhibition of both Kv4.3 and Kv4.3/KChIP2 fast or steady-state inactivation currents with an IC50 of approximately 8 μM. Gliquidone also accelerates Kv4.3 channel inactivation and shifts the steady-state activation to a more depolarizing direction. Site-directed mutagenesis and molecular docking reveal that the residues S301 in the S4 and Y312A and L321A in the S4-S5 linker are critical for gliquidone-mediated inhibition of Kv4.3 currents, as mutating those residues to alanine significantly reduces the potency for gliquidone-mediated inhibition. Furthermore, gliquidone also inhibits a gain-of-function Kv4.3 V392I mutant identified in BrS patients in voltage- and concentration-dependent manner. Taken together, our findings demonstrate that gliquidone inhibits Kv4.3 channels by acting on the residues in the S4 and the S4-S5 linker. Therefore, gliquidone may hold repurposing potential for the therapy of Brugada syndrome. SIGNIFICANCE STATEMENT: We describe a novel role of gliquidone in inhibiting cardiac Kv4.3 currents and the channel gain-of-function mutation identified from patients with Brugada syndrome, suggesting its repurposing potential for therapy for the heart disease.

Differential Effects of the Betablockers Carvedilol, Metoprolol and Bisoprolol on Cardiac Kv4.3 (Ito) Channel Isoforms.

Cardiac Kv4.3 channels contribute to the transient outward K+ current, Ito, during early repolarization of the cardiac action potential. Two different isoforms of Kv4.3 are present in the human ventricle and exhibit differential remodeling in heart failure (HF). Cardioselective betablockers are a cornerstone of HF with reduced ejection fraction therapy as well as ventricular arrhythmia treatment. In this study we examined pharmacological effects of betablockers on both Kv4.3 isoforms to explore their potential for isoform-specific therapy. Kv4.3 isoforms were expressed in Xenopus laevis oocytes and incubated with the respective betablockers. Dose-dependency and biophysical characteristics were examined. HEK 293T-cells were transfected with the two Kv4.3 isoforms and analyzed with Western blots. Carvedilol (100 µM) blocked Kv4.3 L by 77 ± 2% and Kv4.3 S by 67 ± 6%, respectively. Metoprolol (100 µM) was less effective with inhibition of 37 ± 3% (Kv4.3 L) and 35 ± 4% (Kv4.3 S). Bisoprolol showed no inhibitory effect. Current reduction was not caused by changes in Kv4.3 protein expression. Carvedilol inhibited Kv4.3 channels at physiologically relevant concentrations, affecting both isoforms. Metoprolol showed a weaker blocking effect and bisoprolol did not exert an effect on Kv4.3. Blockade of repolarizing Kv4.3 channels by carvedilol and metoprolol extend their pharmacological mechanism of action, potentially contributing beneficial antiarrhythmic effects in normal and failing hearts.

Subtype-specific modulation of human KV 7 channels by the anticonvulsant cannabidiol through a lipid-exposed pore-domain site.

Cannabidiol (CBD) is used clinically as an anticonvulsant. Its precise mechanism of action has remained unclear. CBD was recently demonstrated to enhance the activity of the neuronal KV 7.2/7.3 channel, which may be one important contributor to CBD anticonvulsant effect. Curiously, CBD inhibits the closely related cardiac KV 7.1/KCNE1 channel. Whether and how CBD affects other KV 7 subtypes remains uninvestigated and the CBD interaction sites mediating these diverse effects remain unknown. Here, we used electrophysiology, molecular dynamics simulations, molecular docking and site-directed mutagenesis to address these questions. We found that CBD modulates the activity of all human KV 7 subtypes and that the effects are subtype dependent. CBD enhanced the activity of KV 7.2-7.5 subtypes, seen as a V50 shift towards more negative voltages or increased maximum conductance. In contrast, CBD inhibited the KV 7.1 and KV 7.1/KCNE1 channels, seen as a V50 shift towards more positive voltages and reduced conductance. In KV 7.2 and KV 7.4, we propose a CBD interaction site at the subunit interface in the pore domain that overlaps with the interaction site of other compounds, notably the anticonvulsant retigabine. However, CBD relies on other residues for its effects than the conserved tryptophan that is critical for retigabine effects. We propose a similar, though not identical CBD site in KV 7.1, with a non-conserved phenylalanine being important. We identify novel targets of CBD, contributing to a better understanding of CBD clinical effects and provide mechanistic insights into how CBD modulates different KV 7 subtypes.

Electrophysiological Analysis of the Cardiac KV Channel with the New Point Mutation Found in the Patient with LQTS

Voltage-dependent cation channels Kv7.1 encoded by the KCNQ1 genes play an important role in the repolarization of cardiac cells and the maintenance of normal duration of cardiac action potential. In cardiac muscle tissue, Kv7.1 is expressed together with the accessory KCNE1 beta-subunit to form a functionally active channel that provides a slow repolarization current. To date, more than 100 mutations (mainly single nucleotide variations, SNV) are known to affect the activity of the Kv7.1 channel, altering the kinetics of activation and interaction with regulatory subunits, or disrupting traffic toward the cell membrane.

Correction: Polyunsaturated fatty acid analogues differentially affect cardiac NaV, CaV, and KV channels through unique mechanisms.

The cardiac ventricular action potential depends on several voltage-gated ion channels, including NaV, CaV, and KV channels. Mutations in these channels can cause Long QT Syndrome (LQTS) which increases the risk for ventricular fibrillation and sudden cardiac death. Polyunsaturated fatty acids (PUFAs) have emerged as potential therapeutics for LQTS because they are modulators of voltage-gated ion channels. Here we demonstrate that PUFA analogues vary in their selectivity for human voltage-gated ion channels involved in the ventricular action potential. The effects of specific PUFA analogues range from selective for a specific ion channel to broadly modulating cardiac ion channels from all three families (NaV, CaV, and KV). In addition, a PUFA analogue selective for the cardiac IKs channel (Kv7.1/KCNE1) is effective in shortening the cardiac action potential in human-induced pluripotent stem cell-derived cardiomyocytes. Our data suggest that PUFA analogues could potentially be developed as therapeutics for LQTS and cardiac arrhythmia.

Inhibition of cardiac Kv4.3 (Ito) channel isoforms by class I antiarrhythmic drugs lidocaine and mexiletine

Transient outward K+ current, Ito, contributes to cardiac action potential generation and is primarily carried by Kv4.3 (KCND3) channels. Two Kv4.3 isoforms are expressed in human ventricle and show differential remodeling in heart failure (HF). Lidocaine and mexiletine may be applied in selected patients to suppress ventricular arrhythmias, without effects on sudden cardiac death or mortality. Isoform-dependent effects of antiarrhythmic drugs on Kv4.3 channels and potential implications for remodeling-based antiarrhythmic management have not been assessed to date. We sought to test the hypotheses that Kv4.3 channels are targeted by lidocaine and mexiletine, and that drug sensitivity is determined in isoform-specific manner.Expression of KCND3 isoforms was quantified using qRT-PCR in left ventricular samples of patients with HF due to either ischemic or dilated cardiomyopathies (ICM or DCM). Long (Kv4.3-L) and short (Kv4.3-S) isoforms were heterologously expressed in Xenopus laevis oocytes to study drug sensitivity and effects on biophysical characteristics activation, deactivation, inactivation, and recovery from inactivation.In the present HF patient cohort KCND3 isoform expression did not differ between ICM and DCM. In vitro, lidocaine (IC50-Kv4.3-L: 0.8 mM; IC50-Kv4.3-S: 1.2 mM) and mexiletine (IC50-Kv4.3-L: 146 μM; IC50-Kv4.3-S: 160 μM) inhibited Kv4.3 with different sensitivity. Biophysical analyses identified accelerated and enhanced inactivation combined with delayed recovery from inactivation as primary biophysical mechanisms underlying Kv4.3 current reduction.In conclusion, differential effects on Kv4.3 isoforms extend the electropharmacological profile of lidocaine and mexiletine. Patient-specific remodeling of Kv4.3 isoforms may determine individual drug responses and requires consideration during clinical application of compounds targeting Kv4.3.

Polyunsaturated fatty acid analogues differentially affect cardiac NaV, CaV, and KV channels through unique mechanisms.

The cardiac ventricular action potential depends on several voltage-gated ion channels, including NaV, CaV, and KV channels. Mutations in these channels can cause Long QT Syndrome (LQTS) which increases the risk for ventricular fibrillation and sudden cardiac death. Polyunsaturated fatty acids (PUFAs) have emerged as potential therapeutics for LQTS because they are modulators of voltage-gated ion channels. Here we demonstrate that PUFA analogues vary in their selectivity for human voltage-gated ion channels involved in the ventricular action potential. The effects of specific PUFA analogues range from selective for a specific ion channel to broadly modulating cardiac ion channels from all three families (NaV, CaV, and KV). In addition, a PUFA analogue selective for the cardiac IKs channel (Kv7.1/KCNE1) is effective in shortening the cardiac action potential in human-induced pluripotent stem cell-derived cardiomyocytes. Our data suggest that PUFA analogues could potentially be developed as therapeutics for LQTS and cardiac arrhythmia.

A Novel Treatment for Arrhythmias via the Control of the Degradation of Ion Channel Proteins.

Although there are many reports on the regulation of ion channel expression in transcription and translation, few drugs have been studied to influence post-translational modification of ion channel proteins. The Kv1.5 channel is a potassium ion channel expressed in atrial muscle, belongs to the voltage-gated K+ channel superfamily, and forms an ultrarapid delayed rectifier potassium ion current. It is important to understand the fate of these channel proteins, as cardiac Kv1.5 mutations can cause arrhythmias. Disruption of quantitative and qualitative control mechanisms of channels leads to stagnation and degradation of intracellular channel proteins. As a result, ion channel proteins are not transported to the cell membrane and are involved in the development of atrial fibrillation. This review takes the Kv1.5 channel as an example and focuses on the degradation mechanism of ion channel proteins, and discusses its application to the treatment of arrhythmia by drugs that control the mechanism of ion channel protein degradation.

In vitro and in vivo characterization of a synthetic scorpion toxin AmmTx3, a potent inhibitor of cardiac voltage-gated potassium channel Kv4.2

Voltage-gated potassium channel Kv4.2 (encoded by KCND2 gene) contributes to the cardiac transient outward potassium current (Ito1). This current is the main contributor to the repolarisation phase 1 of the cardiac action potential. The toxin AmmTx3, identified from the venom of the scorpion Androctonus mauretanicus , is a blocker of Kv4.x channels, and have interesting therapeutic potential for neurological disorders due to its effect in cerebellar granule neurons. Its effects on cardiac Kv4.2 channels remains unclear. The aim of this study was to: – produce a synthetic AmmTx3 (AmmTx3-NCL); – evaluate in vitro and in vivo, effects of this toxin on Kv4.2 channels compared to native toxin. We did chemical synthesis of AmmTx3 using the native chemical ligation strategy (NCL). We characterized its biological activity compared to native AmmTx3 using an automated patch-clamp system (Syncropatch 384 PE, Nanion) on HEK cells co-expressing Kv4.2 α subunit and two auxiliaries subunits (KChIP and DPP6). Effects of AmmTx3 and AmmTx3-NCL on cardiac cellular electrophysiology were studied by patch clamp on adult rat cardiomyocytes and hiPSC-CMs. Integrated cardiac effects of AmmTx3 were studied on ECG of wild-type mice. In this study, we report the total chemical synthesis of AmmTx3 and 3D structure was also determined. As expected, biological activity of folded AmmTx3-NCL is coherent to native toxin with a similar time course of Kv4.2 current inhibition without any change of activation kinetics. Characterisation of AmmTx3 effects on isolated cardiomyocytes, hiPSC-CMs and in vivo by ECG is ongoing. AmmTx3 toxin can be chemically synthesized and used as a Kv4.2 channel inhibitor to contributed to the better understanding of the exact role of Ito1 in cardiac electrophysiology. Those first results seem to be a promising evidence that AmmTx3 could a potential inhibitor of Ito current in early repolarisation syndrome.

CaMKII Modulates the Cardiac Transient Outward K+ Current through its Association with Kv4 Channels in Non-Caveolar Membrane Rafts.

To test whether the physiological regulation of the cardiac Kv4 channels by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is restricted to lipid rafts and whether the interactions observed in rat cardiomyocytes also occur in the human ventricle. Ventricular myocytes were freshly isolated from Sprague-Dawley rats. Ito was recorded by the whole-cell Patch-Clamp technique. Membrane rafts were isolated by centrifugation in a discontinuous sucrose density gradient. The presence of the proteins of interest was analysed by western blot. Immunogold staining and electron microscopy of heart vibrosections was performed to localize Kv4.2/Kv4.3 and CaMKII proteins. Protein-protein interactions were determined by co-immunoprecipitation experiments in rat and human ventricular mycoytes. Patch-Clamp recordings in control conditions and after lipid raft or caveolae disruption show that the CaMKII-Kv4 channel complex must associate in non-caveolar lipid rafts to be functional. Separation in density gradients, co-immunoprecipitation and electron microscopy show that there are two Kv4 channel populations: one located in caveolae, that is CaMKII independent, and another one located in planar membrane rafts, which is bound to CaMKII. CaMKII regulates only the Kv4 channel population located in non-caveolar lipid rafts. Thus, the regulation of cardiac Kv4 channels in rat and human ventricle depends on their subcellular localization.

Regulation of human cardiac potassium channels by full-length KCNE3 and KCNE4

Voltage-gated potassium (Kv) channels comprise pore-forming α subunits and a multiplicity of regulatory proteins, including the cardiac-expressed and cardiac arrhythmia-linked transmembrane KCNE subunits. After recently uncovering novel, N-terminally extended (L) KCNE3 and KCNE4 isoforms and detecting their transcripts in human atrium, reported here are their functional effects on human cardiac Kv channel α subunits expressed in Xenopus laevis oocytes. As previously reported for short isoforms KCNE3S and KCNE4S, KCNE3L inhibited hERG; KCNE4L inhibited Kv1.1; neither form regulated the HCN1 pacemaker channel. Unlike KCNE4S, KCNE4L was a potent inhibitor of Kv4.2 and Kv4.3; co-expression of cytosolic β subunit KChIP2, which regulates Kv4 channels in cardiac myocytes, partially relieved Kv4.3 but not Kv4.2 inhibition. Inhibition of Kv4.2 and Kv4.3 by KCNE3L was weaker, and its inhibition of Kv4.2 abolished by KChIP2. KCNE3L and KCNE4L also exhibited subunit-specific effects on Kv4 channel complex inactivation kinetics, voltage dependence and recovery. Further supporting the potential physiological significance of the robust functional effects of KCNE4L on Kv4 channels, KCNE4L protein was detected in human atrium, where it co-localized with Kv4.3. The findings establish functional effects of novel human cardiac-expressed KCNE isoforms and further contribute to our understanding of the potential mechanisms influencing cardiomyocyte repolarization.

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.

Inhibitory effects of hesperetin on Kv1.5 potassium channels stably expressed in HEK 293 cells and ultra-rapid delayed rectifier K+ current in human atrial myocytes

In the present study, the inhibitory effects of hesperetin (HSP) on human cardiac Kv1.5 channels expressed in HEK 293 cells and the ultra-rapid delayed rectifier K+ current (Ikur) in human atrial myocytes were examined by using the whole-cell configuration of the patch-clamp techniques. We found that hesperetin rapidly and reversibly suppressed human Kv1.5 current in a concentration dependent manner with a half-maximal inhibition (IC50) of 23.15 μΜ with a Hill coefficient of 0.89. The current was maximally diminished about 71.36% at a concentration of 300μM hesperetin. Hesperetin significantly positive shifted the steady-state activation curve of Kv1.5, while negative shifted the steady-state inactivation curve. Hesperetin also accelerated the inactivation and markedly slowed the recovery from the inactivation of Kv1.5 currents. Block of Kv1.5 currents by hesperetin was in a frequency dependent manner. However, inclusion of 30μM hesperetin in pipette solution produced no effect on Kv1.5 channel current, while the current were remarkable and reversibly inhibited by extracellular application of 30μM hesperetin. We also found that hesperetin potently and reversibly inhibited the ultra-repaid delayed K+ current (Ikur) in human atrial myocytes, which is in consistent with the effects of hesperetin on Kv1.5 currents in HEK 293 cells. In conclusion, hesperetin is a potent inhibitor of Ikur (which is encoded by Kv1.5), with blockade probably due to blocking of both open state and inactivated state channels from outside of the cell.

HERG S4-S5 acts as a Voltage-Dependent Ligand Binding the Activation Gate and Locking it in a Closed State

Kv (voltage-gated potassium) channels are formed by a tetrameric pore (S5–S6) surrounded by four voltage sensor domains (S1–S4). Molecular mechanisms underlying gating remain poorly understood.Previously, we showed that peptides mimicking S4-S5L inhibit a cardiac Kv channel, KCNQ1 (Kv7.1). On the other hand, peptides mimicking S6T upregulate the channel activity by competing with the endogenous S6T for the endogenous S4-S5L (Choveau et al, 2010). Those observations suggested that S4-S5L acts as a voltage controled ligand that binds S6T and locks the channel in a closed state. However, given the low affinity between the peptide and the KCNQ1 channel, effects were moderate. In the present study, we asked whether covalently locking s4s5l onto s6t would lead to a permanent closure of the channel. To this end, we selected the human ether-a-go-go-related gene (hERG or Kv11.1) channel as a model since in this channel, a couple of cysteine in S4-S5L and S6T was introduced and led to channel closure (Ferrer et al, 2006). We used the whole-cell configuration of the patch-clamp technique in transfected COS-7 cells. We observed a complete inhibition of hERG in oxidative condition when coexpressing the S4-S5L peptide carrying an introduced cysteine (S4S5L-Cys-peptide) and the channel with a cysteine introduced in S6T (S6T-Cys-channel). Reciprocally, coexpressing S6TCyspeptide and the S4-S5L-Cys-channel led to a virtually voltage independent channel in oxidative condition. These results: 1) validate a model of gating in which S4-S5 acts as a voltage-dependent ligand binding the activation gate and locking it in a closed state and 2) suggest a generalization of this model.Choveau et al, J Biol Chem. 2011 286:707-16Ferrer et al, J Biol Chem. 2006 281:12858-64

The inhibitory effects of nifedipine on outward voltage-gated potassium currents in mouse neuroblastoma N2A cells.

Voltage-gated K(+) (Kv) channels have a pivotal role in tuning the action potential duration and excitability in neuronal cells. Although Ca(2+) channel antagonist nifedipine exhibited an inhibitory effect on cardiac Kv currents, a possible action of nifedipine on neuronal Kv currents has not been fully investigated. The effects of nifedipine on elicited Kv currents were characterized using whole-cell recording in mouse neuroblastoma N2A cells. Exposure to nifedipine induced a dose-dependent inhibition of Kv currents with an IC50 value of 22.3±4.2μM and prolonged the time course of activation. The half-maximum activation potential was 1.6±1.7mV in control conditions and became 13.5±1.5mV in 50μM nifedipine. In addition, the decay rate of Kv currents was substantially accelerated by 39.5% at +60mV. For the voltage-dependent inactivation, the half-maximum inactivation potential was -13.8±0.8mV and strongly shifted to the left following treatment with 50μM nifedipine. Treatment with nifedipine exerted a strong influence on the activation and inactivation rate of Kv currents as well as an obvious leftward shift in the inactivation curve. These data indicated that nifedipine exerted an inhibitory effect on Kv currents in N2A cells.

Effects of neferine on Kv4.3 channels expressed in HEK293 cells and ex vivo electrophysiology of rabbit hearts.

Neferine is an isoquinoline alkaloid isolated from seed embryos of Nelumbo nucifera (Gaertn), which has a variety of biological activities. In this study we examined the effects of neferine on Kv4.3 channels, a major contributor to the transient outward current (I(to)) in rabbit heart, and on ex vivo electrophysiology of rabbit hearts. Whole-cell Kv4.3 currents were recorded in HEK293 cells expressing human cardiac Kv4.3 channels using patch-clamp technique. Arterially perfused wedges of rabbit left ventricles (LV) were prepared, and transmembrane action potentials were simultaneously recorded from epicardial (Epi) and endocardial (Endo) sites with floating microelectrodes together with transmural electrocardiography (ECG). Neferine (0.1-100 μmol/L) dose-dependently and reversibly inhibited Kv4.3 currents (the IC50 value was 8.437 μmol/L, and the maximal inhibition at 100 μmol/L was 44.12%). Neferine (10 μmol/L) caused a positive shift of the steady-state activation curve of Kv4.3 currents, and a negative shift of the steady-state inactivation curve. Furthermore, neferine (10 μmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation. Neferine-induced blocking of Kv4.3 currents was frequency-dependent. In arterially perfused wedges of rabbit LV, neferine (1, 3, and 10 μmol/L) dose-dependently prolonged the QT intervals and action potential durations (APD) at both Epi and Endo sites, and caused dramatic increase of APD10 at Epi sites. Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD10 at Epi sites of rabbit heart.

Abstract 14587: Proarrhythmic Effects of Ibrutinib, a Clinically Approved Inhibitor of Bruton’S Tyrosine Kinase (BTK) Used in Cancer Therapy

Introduction: Ibrutinib is a novel and potent BTK inhibitor which has been approved for several types of B-cell malignancies. Our meta-analysis of the published clinical trials finds that atrial fibrillation occurs in ~7% of patients after chronic ibrutinib exposure. Ventricular tachycardia has also been reported. Recently, multi-targeted tyrosine kinase inhibitors (TKIs) have been associated with arrhythmias; one implicated mechanism is increased late sodium current (INa-L) after chronic but not acute drug exposure. This effect appears to be mediated by inhibition of PI3Kinase since it is rescued by intra-pipette PIP3, a PI3K effector. Hypothesis: In this study, we tested the hypothesis that chronic exposure to ibrutinib exerts arrhythmogenic effects by this mechanism. Methods: Isolated adult mouse cardiac atrial/ventricular and rabbit ventricular myocytes as well as KCNH2 (HERG/Kv11.1)- or Kv4.3-transfected CHO cells were used for the electrophysiological studies pre- and post-ibrutinib (0.3 μ or 1 μM) acutely (5-10 min) and chronically (5 hrs in myocytes or 48 hrs in CHO cells). In some experiments, 1 μM PIP3 was added into the pipette solution. Results: Chronic but not acute ibrutinib prolonged cardiac action potentials (APs), triggered abnormal APs (EADs and DADs; Figure), and markedly increased cardiac INa-L (1.68±0.27% vs control 0.21±0.04% of peak current, P<0.01) in mouse atrial cells. A similar INa-L increase was also seen in mouse and rabbit ventricular cells. The abnormal APs and increased INa-L were antagonized by adding 1 μM PIP3 into the pipette and by the INa-L blocker ranolazine (10 μM). Ibrutinib has minor and no effects on cardiac HERG and Kv4.3 currents. Conclusion: Ibrutinib, like other TKIs, increased cardiac INa-L, an effect reversed by intracellular PIP3. These data further reinforce the importance of PIP3 signaling as a proximate mechanism leading to arrhythmogenesis by emerging targeted oncology therapies.

Modulation of the transient outward current (Ito) in rat cardiac myocytes and human Kv4.3 channels by mefloquine

The antimalarial drug mefloquine, is known to be a potassium channel blocker, although its mechanism of action has not being elucidated and its effects on the transient outward current (Ito) and the molecular correlate, the Kv4.3 channel has not being studied. Here, we describe the mefloquine-induced inhibition of the rat ventricular Ito and of CHO cells co-transfected with human Kv4.3 and its accessory subunit hKChIP2C by whole-cell voltage-clamp. Mefloquine inhibited rat Ito and hKv4.3+KChIP2C currents in a concentration-dependent manner with a limited voltage dependence and similar potencies (IC50=8.9μM and 10.5μM for cardiac myocytes and Kv4.3 channels, respectively). In addition, mefloquine did not affect the activation of either current but significantly modified the hKv4.3 steady-state inactivation and recovery from inactivation. The effects of this drug was compared with that of 4-aminopyridine (4-AP), a well-known potassium channel blocker and its binding site does not seem to overlap with that of 4-AP.

Reduced Sialylation Impacts Ventricular Repolarization by Modulating Specific K+ Channel Isoforms Distinctly

Voltage-gated K(+) channels (Kv) are responsible for repolarizing excitable cells and can be heavily glycosylated. Cardiac Kv activity is indispensable where even minimal reductions in function can extend action potential duration, prolong QT intervals, and ultimately contribute to life-threatening arrhythmias. Diseases such as congenital disorders of glycosylation often cause significant cardiac phenotypes that can include arrhythmias. Here we investigated the impact of reduced sialylation on ventricular repolarization through gene deletion of the sialyltransferase ST3Gal4. ST3Gal4-deficient mice (ST3Gal4(-/-)) had prolonged QT intervals with a concomitant increase in ventricular action potential duration. Ventricular apex myocytes isolated from ST3Gal4(-/-) mice demonstrated depolarizing shifts in activation gating of the transient outward (Ito) and delayed rectifier (IKslow) components of K(+) current with no change in maximum current densities. Consistently, similar protein expression levels of the three Kv isoforms responsible for Ito and IKslow were measured for ST3Gal4(-/-) versus controls. However, novel non-enzymatic sialic acid labeling indicated a reduction in sialylation of ST3Gal4(-/-) ventricular Kv4.2 and Kv1.5, which contribute to Ito and IKslow, respectively. Thus, we describe here a novel form of regulating cardiac function through the activities of a specific glycogene product. Namely, reduced ST3Gal4 activity leads to a loss of isoform-specific Kv sialylation and function, thereby limiting Kv activity during the action potential and decreasing repolarization rate, which likely contributes to prolonged ventricular repolarization. These studies elucidate a novel role for individual glycogene products in contributing to a complex network of cardiac regulation under normal and pathologic conditions.