Action Potential Repolarization Research Articles

An Eag Domain Polypeptide Regulates the Deactivation Kinetics of the hERG 1A-3.1 Splice Variant Linked to Schizophrenia

The Human Ether-a-go-go Related Gene (hERG) encodes a voltage-activated K+ channel. hERG contributes to the repolarization of the ventricular action potential as the primary component of the cardiac delayed rectifier K+ current (IKr) and has also been shown by many groups to modulate neuronal firing frequency. Disruption of channel function by inherited mutations in the gene encoding hERG has been shown to cause type 2 long QT syndrome (LQT2). hERG gating is characterized by rapid inactivation upon depolarization and rapid recovery from inactivation and slow closing (deactivation) upon repolarization. Unlike other K+ channels, hERG, ELK and EAG channels contain an N-terminal eag domain, composed of a PAS domain and a PAS-Cap domain in the N-terminal region. Inter-subunit interactions between the N-terminal eag domain and the C-terminal cyclic-nucleotide binding homology domain regulate slow deactivation kinetics in the original variant of hERG, hERG1a channels. A recently discovered splice variant, hERG 1a-3.1, has much faster deactivation and has been linked to schizophrenia. We demonstrate in HEK293 cells, that co-expression of hERG1a-3.1 with soluble eag domain peptide partially rescues deactivation kinetics, presumably by providing a fully functional eag domain to the eag-truncated hERG1a-3.1 to slow channel deactivation. Cells co-expressing hERG1a-3.1 + eag domain polypeptides had significantly slower fast and slow time constants of deactivation compared to hERG1a-3.1 channels alone (p<0.05). We conclude that eag domain polypeptides are able to partially restore deactivation gating. Current experiments are utilizing FRET to assess if the hERG1a-3.1 C-terminus interaction with the eag domain is physically disrupted.

Intracellular Calcium Alters IKs Amplitude and Kinetics in Rabbit Myocytes

The slowly activating delayed rectifier K+ current (IKs) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca2+ ([Ca2+]i) can modulate IKs, but details of this potentially important regulation are largely unknown. Here, we aimed at assessing [Ca2+]i regulation of IKs and how it governs myocyte AP duration. IKs was recorded from freshly isolated rabbit ventricular myocytes using the patch clamp technique with intracellular pipette solutions that buffered [Ca2+]i to 0, 100, 300, and 500 nM. From a holding potential of −50 mV, IKs was recorded by implementing step-like pulses from −40 to 50 mV in 10 mV increments for 3 s, followed by a tail-pulse to −50 mV for 3 s. When the [Ca2+]i was increased to 300 nM, the maximally activated tail IKs (IMAX) was more than 2-fold greater compared to 0 nM [Ca2+]i (IMAX ∼0.8 pA/pF vs. 0.3 pA/pF). Importantly, when the pipette solution contained 500 nM [Ca2+]i (IMAX ∼1.0 pA/pF), IMAX was 3-fold greater than 0 nM and more than 2-fold greater than 100 nM [Ca2+]i (IMAX ∼0.4 pA/pF). The potential of half-maximal activation (V½) was not different for any situations (∼15 mV). However, deactivation kinetics of tail IKs were slower for cells recorded with 300 and 500 nM [Ca2+]i compared to 0 and 100 nM [Ca2+]i (τdeact ∼1200 ms vs. 800 ms). These results indicate that a rise in [Ca2+]i increases IKs amplitude in rabbits, without altering the voltage dependence of activation, and slows IKs deactivation. Computational modeling suggests these [Ca2+]i-dependent changes might contribute to ventricular AP duration alterations, especially in the presence of adrenergic activation.

Stoichiometry of a hERG1 Agonist on Channel Gating

Human ether-a-go-go-related gene 1 (hERG1) K+ channels mediate repolarization of action potentials in cardiomyocytes. A refined mechanistic understanding of the action of hERG1 agonists may assist development of novel compounds for treatment of arrhythmia associated with acquired or congenital long QT syndrome. RPR-260243 [(3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5 trifluorophenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid] (RPR) profoundly slows deactivation and modestly attenuates fast C-type inactivation of hERG1 channels without affecting its kinetics. A Markov model of channel gating incorporating two open states reproduced these experimental findings. A binding site for RPR has been localized to a hydrophobic pocket between two adjacent hERG1 subunits. Accordingly, one homotetrameric hERG1 channel contains four identical RPR binding sites. In this study, the stoichiometric basis of RPR-altered hERG1 channel currents was investigated. Concatenated hERG1 tetramers incorporating a variable number of wild-type subunits and mutant subunits (L553A to prevent RPR binding) were constructed and heterologously expressed in Xenopus oocytes. RPR slowed the rate of hERG1 channel deactivation as a function of the number of wild-type subunits (i.e., accessible RPR binding sites) contained within a tetramer. Occupancy of all four available binding sites was required to attain maximum effect. In contrast, occupancy of only three binding sites was sufficient to achieve the maximum shift (+24 mV) in the voltage required for half-maximal inactivation. The distinct subunit stoichiometries associated with RPR-induced changes in hERG1 deactivation and inactivation reflects the different structural basis of the activation (S6 bundle crossing) and inactivation (selectivity filter) gates.

Effect of Amitriptyline in Kv7.1/MinK Channel

The KCNQ1 gene encodes the voltage-gated K+ channel Kv7.1, which is mainly expressed in cardiac muscle. Coassembly with the β-subunit (MinK) Kv7.1 generates a very slowly activating delayed-rectifier K+ current, IKs, with no apparent inactivation.It has been reported that amitriptyline, a tricyclic antidepressant, inhibits the Kv1.1 and Kv7.2/7.3 K+ channels in a voltage-independent but concentration-dependent manner. However, there is no evidence of the effect of this drug on Kv7.1; a channel of the same K+ channel family than Kv7.2/Kv7.3 but with different kinetics and sequential characteristics.Amitriptyline has been shown to induce long QT syndrome and torsades de pointes in human hearts which cause sudden death. This effect was related to HERG channel blockage, the molecular correlate of the rapid activated delayed rectifier K+ current (IKr); however, the drug effects on IKs, a major determinant of action potential repolarization in the heart, has not been studied yet.In this study we show that amitriptyline inhibits Kv7.1/MinK in a concentration-dependent manner with an IC50 of 3.27 µM. Inhibition of these channels was voltage-independent and reversible. The voltage dependence activation of the channel was not modified by amitriptyline. We assessed the effect of the drug on Kv7.1 channels assembled as homotetramer, without the accessory subunit. Kv7.1 channel current was less sensitive to inhibition by amitriptyline (IC50 13.17 µM) than heteromeric Kv7.1/MinK channel current but like Kv7.1/MinK, current inhibition was voltage independent. Our results demonstrate that amitriptyline inhibits Kv7.1/MinK channels and we suggest that the drug acts on the pore forming subunit Kv7.1 instead of MinK.

Dominant Negative Consequences of a HERG 1B Mutation Associated with Intrauterine Fetal Death

The human ether-a-go-go related gene (hERG) encodes the voltage-gated potassium channel responsible for conducting the rapid delayed rectifier potassium current (IKr). Reduced IKr slows cardiac action potential repolarization and is an underlying cause of cardiac arrhythmias associated with both inherited and acquired long QT syndrome (LQTS). Two subunits comprise the hERG channel, hERG 1a and hERG 1b, both of which represent critical components of cardiac repolarization. R25W is a hERG 1b mutation associated with a case of intrauterine fetal death and previously shown to reduce heteromeric hERG 1a/1b current density in Chinese hamster ovarian cells1. The mechanism of current reduction and a direct link to cardiac dysfunction has not been described for R25W. We expressed R25W in HEK cells and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to define the molecular, biophysical, and physiological consequences of R25W. R25W significantly reduced 1a and 1b protein levels to 76.4±6.0% and 65.2±7.8% of control, respectively, in HEK cells coexpressing hERG 1a and 1b. In iPSC-CMs, R25W transfection significantly reduced peak tail IKr (0.8±0.1 pA/pF, n = 10, p < 0.05) compared to either native IKr (1.7±0.2 pA/pF, n = 5) or wild type 1b transfected iPSC-CMs (1.5±0.3 pA/pF, n = 7). Interestingly, wild-type 1b did not affect peak tail IKr whereas transfection of either 1a alone (35.9 ± 11.0 pA/pF, n = 5) or 1a/1b together (24.8±11.2 pA/pF, n = 5) dramatically increased current density. These data identify R25W as a dominant negative mutation that reduces native IKr and provide new evidence that hERG 1b fails to traffic as a homomeric channel in human cardiomyocytes. Supported by NIH/R01HL081780 and the UW Training Program in Translational Cardiovascular Science (T32HL007936). 1. Crotti et al., 2013, JAMA.

Probing the Trafficking Routes of KCNQ1 and KCNE1 After Their ER Exit

Background: KCNQ1 (Q1, pore-forming channel subunit) and KCNE1 (E1, regulatory subunit) associate to form the slow delayed rectifier (IKs) channel, important for cardiac action potential repolarization. Although Q1/E1 are obligatory partners in cardiomyocytes, in adult ventricular myocytes the two are not well colocalized: E1 is on the lateral cell surface while Q1 is mainly in the intracellular junctional SR. This appears to contradict data from heterologous expression or non-cardiac cell types, which suggest that Q1/E1 assemble into IKs when they are still in ER or soon thereafter. The current study is designed to distinguish between 2 scenarios. First, Q1 and E1 traffic from ER to the plasma membrane together, followed by Q1 internalization to the ER/SR compartment. Alternatively, Q1 and E1 traffic on different routes from ER to their separate destinations.Methods: COS-7 cells transfected with Q-GFP/E1-dsR are cultured in presence of brefeldin A (BFA) for 12 – 16 hr, to allow protein translation without ER exit. Cells are imaged in absence of BFA but presence of cycloheximide+dynasore (blocking protein translation and endocytosis). Time-lapse images (Zeiss 710, 37oC) of 4-um optic slice (to maximize capture of trafficking events in thin cytosol of COS-7 cells) are recorded till the Q1-GFP and E1-dsR distribution reaches quasi steady-state (> 2 hr).Results: BFA removal allows ER exit and Golgi reconstruction. E1-dsR exits ER in distinct vesicles, more frequently at the cell periphery. These vesicles gradually cluster to the peri-nuclear region, entering the post-BFA Golgi. Q1-QFP stays in the ER compartment for > 2 hr. Its ER location is confirmed by FRAP experiments.Conclusion: Our data support the second scenario. Experiments are in progress to test whether E1-dsR and Q1-GFP behave the same when expressed in a cardiac myocyte environment.

Role of gap junction channel in the development of beat-to-beat action potential repolarization variability and arrhythmias.

The short-term beat-to-beat variability of cardiac action potential duration (SBVR) occurs as a random alteration of the ventricular repolarization duration. SBVR has been suggested to be more predictive of the development of lethal arrhythmias than the action potential prolongation or QT prolongation of ECG alone. The mechanism underlying SBVR is not completely understood but it is known that SBVR depends on stochastic ion channel gating, intracellular calcium handling and intercellular coupling. Coupling of single cardiomyocytes significantly decreases the beat-to-beat changes in action potential duration (APD) due to the electrotonic current flow between neighboring cells. The magnitude of this electrotonic current depends on the intercellular gap junction resistance. Reduced gap junction resistance causes greater electrotonic current flow between cells, and reduces SBVR. Myocardial ischaemia (MI) is known to affect gap junction channel protein expression and function. MI increases gap junction resistance that leads to slow conduction, APD and refractory period dispersion, and an increase in SBVR. Ultimately, development of reentry arrhythmias and fibrillation are associated post-MI. Antiarrhythmic drugs have proarrhythmic side effects requiring alternative approaches. A novel idea is to target gap junction channels. Specifically, the use of gap junction channel enhancers and inhibitors may help to reveal the precise role of gap junctions in the development of arrhythmias. Since cell-to-cell coupling is represented in SBVR, this parameter can be used to monitor the degree of coupling of myocardium.

Concatenated hERG1 tetramers reveal stoichiometry of altered channel gating by RPR-260243.

Activation of human ether-a-go-go-related gene 1 (hERG1) K(+) channels mediates repolarization of action potentials in cardiomyocytes. RPR-260243 [(3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluorophenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid] (RPR) slows deactivation and attenuates inactivation of hERG1 channels. A detailed understanding of the molecular mechanism of hERG1 agonists such as RPR may facilitate the design of more selective and potent compounds for prevention of arrhythmia associated with abnormally prolonged ventricular repolarization. RPR binds to a hydrophobic pocket located between two adjacent hERG1 subunits, and, hence, a homotetrameric channel has four identical RPR binding sites. To investigate the stoichiometry of altered channel gating induced by RPR, we constructed and characterized tetrameric hERG1 concatemers containing a variable number of wild-type subunits and subunits containing a point mutation (L553A) that rendered the channel insensitive to RPR, ostensibly by preventing ligand binding. The slowing of deactivation by RPR was proportional to the number of wild-type subunits incorporated into a concatenated tetrameric channel, and four wild-type subunits were required to achieve maximal slowing of deactivation. In contrast, a single wild-type subunit within a concatenated tetramer was sufficient to achieve half of the maximal RPR-induced shift in the voltage dependence of hERG1 inactivation, and maximal effect was achieved in channels containing three or four wild-type subunits. Together our findings suggest that the allosteric modulation of channel gating involves distinct mechanisms of coupling between drug binding and altered deactivation and inactivation.

HERG 1b is critical for human cardiac repolarization.

The human ether-à-go-go-related gene (hERG; or KCNH2) encodes the voltage-gated potassium channel underlying IKr, a repolarizing current in the heart. Mutations in KCNH2 or pharmacological agents that reduce IKr slow action potential (AP) repolarization and can trigger cardiac arrhythmias associated with long QT syndrome. Two channel-forming subunits encoded by KCNH2 (hERG 1a and 1b) are expressed in cardiac tissue. In heterologous expression systems, these subunits avidly coassemble and exhibit biophysical and pharmacological properties distinct from those of homomeric hERG 1a channels. Despite these findings, adoption of hERG 1a/1b heteromeric channels as a model for cardiac IKr has been hampered by the lack of evidence for a direct functional role for the 1b subunit in native tissue. In this study, we measured IKr and APs at physiological temperature in cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs). We found that specific knockdown of the 1b subunit using shRNA caused reductions in 1b mRNA, 1b protein levels, and IKr magnitude by roughly one-half. AP duration was increased and AP variability was enhanced relative to controls. Early afterdepolarizations, considered cellular substrates for arrhythmia, were also observed in cells with reduced 1b expression. Similar behavior was elicited when channels were effectively converted from heteromers to 1a homomers by expressing a fragment corresponding to the 1a-specific N-terminal Per-Arnt-Sim domain, which is omitted from hERG 1b by alternate transcription. These findings establish that hERG 1b is critical for normal repolarization and that loss of 1b is proarrhythmic in human cardiac cells.

TASK-1 current is inhibited by phosphorylation during human and canine chronic atrial fibrillation.

Atrial fibrillation (AF) is a common arrhythmia with significant morbidities and only partially adequate therapeutic options. AF is associated with atrial remodeling processes, including changes in the expression and function of ion channels and signaling pathways. TWIK protein-related acid-sensitive K+ channel (TASK)-1, a two-pore domain K+ channel, has been shown to contribute to action potential repolarization as well as to the maintenance of resting membrane potential in isolated myocytes, and TASK-1 inhibition has been associated with the induction of perioperative AF. However, the role of TASK-1 in chronic AF is unknown. The present study investigated the function, expression, and phosphorylation of TASK-1 in chronic AF in atrial tissue from chronically paced canines and in human subjects. TASK-1 current was present in atrial myocytes isolated from human and canine hearts in normal sinus rhythm but was absent in myocytes from humans with AF and in canines after the induction of AF by chronic tachypacing. The addition of phosphatase to the patch pipette rescued TASK-1 current from myocytes isolated from AF hearts, indicating that the change in current is phosphorylation dependent. Western blot analysis showed that total TASK-1 protein levels either did not change or increased slightly in AF, despite the absence of current. In studies of perioperative AF, we have shown that phosphorylation of TASK-1 at Thr383 inhibits the channel. However, phosphorylation at this site was unchanged in atrial tissue from humans with AF or in canines with chronic pacing-induced AF. We conclude that phosphorylation-dependent inhibition of TASK-1 is associated with AF, but the phosphorylation site responsible for this inhibition remains to be identified.

Abstract 17272: Ionic Current Remodeling Delays the Electrical Recovery of the Senescent Heart

Cardiac aging is associated with lengthening of the QT interval, a condition that enhances malignant ventricular arrhythmias and sudden death. The aim of this study was to establish whether ionic currents are altered in old myocytes contributing to the protracted electrical recovery of the senescent heart. Thus, mice at 3-30 months of age were studied by ECG and patch-clamp; these physiological determinations were complemented with molecular assays for the analysis of ion channel proteins. By surface ECG and telemetry system, PR, QRS and QT intervals were prolonged in mice at 25 months or older. These delays were maintained in ex-vivo Langendorff preparations. In comparison to young, epicardial monophasic action potential (AP) duration at 50% and 90% repolarization were 1.6- and 1.2-fold larger in old LV, respectively. Moreover, senescent hearts presented a 60% higher incidence of arrhythmias. In isolated myocytes, prolongation of the early (+47%), intermediate (+117%) and late (+75%) repolarization phases of the AP were identified in cells from old animals, whereas resting membrane potential, upstroke amplitude and +dV/dt were preserved. Voltage-clamp experiments were then performed to measure ionic current properties. The rapidly activating K+ current, which consists of the transient outward and ultrarapid delayed rectifier (Ito+Kur), is responsible for the early repolarization of the AP, and was significantly reduced in old myocytes. Molecular studies revealed low levels of transcripts and proteins for K+ channel subunits Kv1.4, Kv1.5 and KChiP2 in senescent cells. Also, the late Na+ current INaL, which presents slow inactivation kinetics and is operative during AP repolarization, was 1.5-fold larger in old cells. These changes were associated with alterations in gene and protein expression of Na+ channel subunits. Inhibition of INaL with mexiletine significantly shortened the intermediate and late repolarization phases of the AP in both myocytes and perfused myocardium from old mice. Importantly, INaL inhibition in vivo shortened the QT interval of senescent mice by 12%. Thus, defects in ionic current occur with aging resulting in prolongation of the AP and delays in electrical recovery which may lead to malignant ventricular arrhythmias.

Components of action potential repolarization in cerebellar parallel fibres.

Repolarization of the presynaptic action potential is essential for transmitter release, excitability and energy expenditure. Little is known about repolarization in thin, unmyelinated axons forming en passant synapses, which represent the most common type of axons in the mammalian brain's grey matter.We used rat cerebellar parallel fibres, an example of typical grey matter axons, to investigate the effects of K(+) channel blockers on repolarization. We show that repolarization is composed of a fast tetraethylammonium (TEA)-sensitive component, determining the width and amplitude of the spike, and a slow margatoxin (MgTX)-sensitive depolarized after-potential (DAP). These two components could be recorded at the granule cell soma as antidromic action potentials and from the axons with a newly developed miniaturized grease-gap method. A considerable proportion of fast repolarization remained in the presence of TEA, MgTX, or both. This residual was abolished by the addition of quinine. The importance of proper control of fast repolarization was demonstrated by somatic recordings of antidromic action potentials. In these experiments, the relatively broad K(+) channel blocker 4-aminopyridine reduced the fast repolarization, resulting in bursts of action potentials forming on top of the DAP. We conclude that repolarization of the action potential in parallel fibres is supported by at least three groups of K(+) channels. Differences in their temporal profiles allow relatively independent control of the spike and the DAP, whereas overlap of their temporal profiles provides robust control of axonal bursting properties.

Dynamic excitation states and firing patterns are controlled by sodium channel kinetics in myenteric neurons: a simulation study

Enteric neurons located in the gastro-intestinal tract are of particular importance to control digestive functions such as motility and secretion. In our recent publication, we showed that mouse myenteric neurons exhibit 2 types of tetrodotoxin-resistant Na+ currents: a fast inactivating Na+ current produced by Nav1.5 channels, present in nearly all myenteric neurons, and a persistent Na+ current attributed to Nav1.9 channels, restricted to the intrinsic primary afferent neurons (IPANs). By combination of experimental recording and computer simulation we found that Nav1.5 contributed to the upstroke velocity of action potentials (APs), whereas Nav1.9 opposed AP repolarization. Here, we detailed the Na+, Ca2+ and K+ currents used in our computational model of IPAN. We refined the prototype cell to reproduce the sustained firing pattern recorded in situ. As shown in experimental conditions we demonstrated that Nav1.9 channels critically determine the up-state life-time and thus, are essential to sustain tonic firing.

Electrical Excitation of the Heart in a Basal Vertebrate, the European River Lamprey (Lampetra fluviatilis)

Hagfishes and lampreys (order Cyclostomata) are living representatives of an ancient group of jawless vertebrates (class Agnatha). Studies on cyclostome hearts may provide insights into the evolution of the vertebrate heart and thereby increase our understanding of cardiac function in higher vertebrates, including mammals. To this end, electrical excitability of the heart in a basal vertebrate, the European river lamprey (Lampetra fluviatilis), was examined. Ion currents of cardiac myocytes, action potentials (APs) of atrial and ventricular muscle, and electrocardiogram (in vivo) were measured using the patch-clamp method, intracellular microelectrodes, and trailing wires, respectively. The characteristic features of fairly high heart rate (28.4 ± 3 beats min(-1)) and short AP duration (550 ± 44 and 122.1 ± 28.5 for ventricle and atrium, respectively) at low ambient temperature (5°C) are shared with cold-active teleost fishes. However, the ion current basis of the ventricular AP differs from that of other fishes. For inward currents, sodium current density (INa) is lower and calcium current density (ICa) higher than in teleost ventricles, while the kinetics of INa is slow and that of ICa is fast in comparison. Among the ventricular repolarizing currents, the delayed rectifier K(+) current is smaller than in myocytes of several teleost species. Unlike mammalian hearts, ATP-sensitive K(+) channels are constitutively open under normoxic conditions, thus contributing to negative resting membrane potential and repolarization of APs. Upstroke velocity of AP (5.4 ± 0.9 and 6.3 ± 0.6 V s(-1) for ventricular and atrial myocytes, respectively) is slower than in teleost hearts. Excitability of the lamprey heart seems to possess both primitive and advanced characteristics. Short APs are appropriate to support brief and vigorous contractions (in common with higher vertebrates), while relatively low AP upstroke velocities enable only relatively slow propagation of contraction over the heart.

Archaerhodopsin voltage imaging: synaptic calcium and BK channels stabilize action potential repolarization at the Drosophila neuromuscular junction.

The strength and dynamics of synaptic transmission are determined, in part, by the presynaptic action potential (AP) waveform at the nerve terminal. The ion channels that shape the synaptic AP waveform remain essentially unknown for all but a few large synapses amenable to electrophysiological interrogation. The Drosophila neuromuscular junction (NMJ) is a powerful system for studying synaptic biology, but it is not amenable to presynaptic electrophysiology. Here, we demonstrate that Archaerhodopsin can be used to quantitatively image AP waveforms at the Drosophila NMJ without disrupting baseline synaptic transmission or neuromuscular development. It is established that Shaker mutations cause a dramatic increase in neurotransmitter release, suggesting that Shaker is predominantly responsible for AP repolarization. Here we demonstrate that this effect is caused by a concomitant loss of both Shaker and slowpoke (slo) channel activity because of the low extracellular calcium concentrations (0.2-0.5 mM) used typically to assess synaptic transmission in Shaker. In contrast, at physiological extracellular calcium (1.5 mM), the role of Shaker during AP repolarization is limited. We then provide evidence that calcium influx through synaptic CaV2.1 channels and subsequent recruitment of Slo channel activity is important, in concert with Shaker, to ensure proper AP repolarization. Finally, we show that Slo assumes a dominant repolarizing role during repetitive nerve stimulation. During repetitive stimulation, Slo effectively compensates for Shaker channel inactivation, stabilizing AP repolarization and limiting neurotransmitter release. Thus, we have defined an essential role for Slo channels during synaptic AP repolarization and have revised our understanding of Shaker channels at this model synapse.

Characterization of Cardiac Anoctamin1 Ca²⁺-Activated Chloride Channels and Functional Role in Ischemia-Induced Arrhythmias.

Anoctamin1 (ANO1) encodes a Ca(2+)-activated chloride (Cl(-)) channel (CaCC) in variety tissues of many species. Whether ANO1 expresses and functions as a CaCC in cardiomyocytes remain unknown. The objective of this study is to characterize the molecular and functional expression of ANO1 in cardiac myocytes and the role of ANO1-encoded CaCCs in ischemia-induced arrhythmias in the heart. Quantitative real-time RT-PCR, immunofluorescence staining assays, and immunohistochemistry identified the molecular expression, location, and distribution of ANO1 in mouse ventricular myocytes (mVMs). Patch-clamp recordings combined with pharmacological analyses found that ANO1 was responsible for a Ca(2+)-activated Cl(-) current (I(Cl.Ca)) in cardiomyocytes. Myocardial ischemia led to a significant increase in the current density of I(Cl.Ca), which was inhibited by a specific ANO1 inhibitor, T16A(inh)-A01, and an antibody targeting at the pore area of ANO1. Moreover, cardiomyocytes isolated from mice with ischemia-induced arrhythmias had an accelerated early phase 1 repolarization of action potentials (APs) and a deeper "spike and dome" compared to control cardiomyocytes from non-ischemia mice. Application of the antibody targeting at ANO1 pore prevented the ischemia-induced early phase 1 repolarization acceleration and caused a much shallower "spike and dome". We conclude that ANO1 encodes CaCC and plays a significant role in the phase 1 repolarization of APs in mVMs. The ischemia-induced increase in ANO1 expression may be responsible for the increased density of I(Cl.Ca) in the ischemic heart and may contribute, at least in part, to ischemia-induced arrhythmias.

Calcium-activated potassium current modulates ventricular repolarization in chronic heart failure.

The role of IKCa in cardiac repolarization remains controversial and varies across species. The relevance of the current as a therapeutic target is therefore undefined. We examined the cellular electrophysiologic effects of IKCa blockade in controls, chronic heart failure (HF) and HF with sustained atrial fibrillation. We used perforated patch action potential recordings to maintain intrinsic calcium cycling. The IKCa blocker (apamin 100 nM) was used to examine the role of the current in atrial and ventricular myocytes. A canine tachypacing induced model of HF (1 and 4 months, n = 5 per group) was used, and compared to a group of 4 month HF with 6 weeks of superimposed atrial fibrillation (n = 7). A group of age-matched canine controls were used (n = 8). Human atrial and ventricular myocytes were isolated from explanted end-stage failing hearts which were obtained from transplant recipients, and studied in parallel. Atrial myocyte action potentials were unchanged by IKCa blockade in all of the groups studied. IKCa blockade did not affect ventricular myocyte repolarization in controls. HF caused prolongation of ventricular myocyte action potential repolarization. IKCa blockade caused further prolongation of ventricular repolarization in HF and also caused repolarization instability and early afterdepolarizations. SK2 and SK3 expression in the atria and SK3 in the ventricle were increased in canine heart failure. We conclude that during HF, IKCa blockade in ventricular myocytes results in cellular arrhythmias. Furthermore, our data suggest an important role for IKCa in the maintenance of ventricular repolarization stability during chronic heart failure. Our findings suggest that novel antiarrhythmic therapies should have safety and efficacy evaluated in both atria and ventricles.

Knockout of the BK β2 subunit abolishes inactivation of BK currents in mouse adrenal chromaffin cells and results in slow-wave burst activity

Rat and mouse adrenal medullary chromaffin cells (CCs) express an inactivating BK current. This inactivation is thought to arise from the assembly of up to four β2 auxiliary subunits (encoded by the kcnmb2 gene) with a tetramer of pore-forming Slo1 α subunits. Although the physiological consequences of inactivation remain unclear, differences in depolarization-evoked firing among CCs have been proposed to arise from the ability of β2 subunits to shift the range of BK channel activation. To investigate the role of BK channels containing β2 subunits, we generated mice in which the gene encoding β2 was deleted (β2 knockout [KO]). Comparison of proteins from wild-type (WT) and β2 KO mice allowed unambiguous demonstration of the presence of β2 subunit in various tissues and its coassembly with the Slo1 α subunit. We compared current properties and cell firing properties of WT and β2 KO CCs in slices and found that β2 KO abolished inactivation, slowed action potential (AP) repolarization, and, during constant current injection, decreased AP firing. These results support the idea that the β2-mediated shift of the BK channel activation range affects repetitive firing and AP properties. Unexpectedly, CCs from β2 KO mice show an increased tendency toward spontaneous burst firing, suggesting that the particular properties of BK channels in the absence of β2 subunits may predispose to burst firing.

Andersen-Tawil syndrome: a review of its clinical and genetic diagnosis with emphasis on cardiac manifestations

The Andersen-Tawil syndrome is a cardiac ion channel disease that is inherited in an autosomal dominant way and is classified as type 7 of the congenital long QT syndromes. Affected gene is KCNJ2, which forms the inward rectifier potassium channel designated Kir2.1. This protein is involved in stabilizing the resting membrane potential and controls the duration of the action potential in skeletal muscle and heart. It also participates in the terminal repolarization phase of the action potential in ventricular myocytes and is a major component responsible for the correction in the potassium current during phase 3 of the action potential repolarization. Kir 2.1 channel has a predominant role in skeletal muscle, heart and brain. Alterations in this channel produce flaccid paralysis, arrhythmias, impaired skeletal development primarily in extremities and facial area. In this review we address the disease from the point of view of clinical and molecular diagnosis with emphasis on cardiac manifestations.

Nonequilibrium reactivation of Na+ current drives early afterdepolarizations in mouse ventricle.

Early afterdepolarizations (EADs) are triggers of cardiac arrhythmia driven by L-type Ca(2+) current (ICaL) reactivation or sarcoplasmic reticulum Ca(2+) release and Na(+)/Ca(2+) exchange. In large mammals the positive action potential plateau promotes ICaL reactivation, and the current paradigm holds that cardiac EAD dynamics are dominated by interaction between ICaL and the repolarizing K(+) currents. However, EADs are also frequent in the rapidly repolarizing mouse action potential, which should not readily permit ICaL reactivation. This suggests that murine EADs exhibit unique dynamics, which are key for interpreting arrhythmia mechanisms in this ubiquitous model organism. We investigated these dynamics in myocytes from arrhythmia-susceptible calcium calmodulin-dependent protein kinase II delta C (CaMKIIδC)-overexpressing mice (Tg), and via computational simulations. In Tg myocytes, β-adrenergic challenge slowed late repolarization, potentiated sarcoplasmic reticulum Ca(2+) release, and initiated EADs below the ICaL activation range (-47 ± 0.7 mV). These EADs were abolished by caffeine and tetrodotoxin (but not ranolazine), suggesting that sarcoplasmic reticulum Ca(2+) release and Na(+) current (INa), but not late INa, are required for EAD initiation. Simulations suggest that potentiated sarcoplasmic reticulum Ca(2+) release and Na(+)/Ca(2+) exchange shape late action potential repolarization to favor nonequilibrium reactivation of INa and thereby drive the EAD upstroke. Action potential clamp experiments suggest that lidocaine eliminates virtually all inward current elicited by EADs, and that this effect occurs at concentrations (40-60 μmol/L) for which lidocaine remains specific for inactivated Na(+) channels. This strongly suggests that previously inactive channels are recruited during the EAD upstroke, and that nonequilibrium INa dynamics underlie murine EADs. Nonequilibrium reactivation of INa drives murine EADs.