Kirstine Calloe

A transient outward potassium current activator recapitulates the electrocardiographic manifestations of Brugada syndrome

AIMS Transient outward potassium current (I(to)) is thought to be central to the pathogenesis of the Brugada syndrome (BrS). However, an I((to)) activator has not been available with which to validate this hypothesis. Here, we provide a direct test of the hypothesis using a novel I(to) activator, NS5806. METHODS AND RESULTS Isolated canine ventricular myocytes and coronary-perfused wedge preparations were used. Whole-cell patch-clamp studies showed that NS5806 (10 microM) increased peak I(to) at +40 mV by 79 +/- 4% (24.5 +/- 2.2 to 43.6 +/- 3.4 pA/pF, n = 7) and slowed the time constant of inactivation from 12.6 +/- 3.2 to 20.3 +/- 2.9 ms (n = 7). The total charge carried by I(to) increased by 186% (from 363.9 +/- 40.0 to 1042.0 +/- 103.5 pA x ms/pF, n = 7). In ventricular wedge preparations, NS5806 increased phase 1 and notch amplitude of the action potential in the epicardium, but not in the endocardium, and accentuated the ECG J-wave, leading to the development of phase 2 re-entry and polymorphic ventricular tachycardia (n = 9). Although sodium and calcium channel blockers are capable of inducing BrS only in right ventricular (RV) wedge preparations, the I(to) activator was able to induce the phenotype in wedges from both ventricles. NS5806 induced BrS in 4/6 right and 2/10 left ventricular wedge preparations. CONCLUSION The I(to) activator NS5806 recapitulates the electrographic and arrhythmic manifestation of BrS, providing evidence in support of its pivotal role in the genesis of the disease. Our findings also suggest that a genetic defect leading to a gain of function of I(to) could explain variants of BrS, in which ST-segment elevation or J-waves are evident in both right and left ECG leads.

The Small Molecule NS11021 Is a Potent and Specific Activator of Ca2+-Activated Big-Conductance K+ Channels

Large-conductance Ca2+- and voltage-activated K+ channels (Kca1.1/BK/MaxiK) are widely expressed ion channels. They provide a Ca2+-dependent feedback mechanism for the regulation of various body functions such as blood flow, neurotransmitter release, uresis, and immunity. In addition, a mitochondrial K+ channel with KCa1.1-resembling properties has been found in the heart, where it may be involved in regulation of energy consumption. In the present study, the effect of a novel NeuroSearch compound, 1-(3,5-bis-trifluoromethyl-phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea (NS11021), was investigated on cloned KCa1.1 expressed in Xenopus laevis oocytes and mammalian cells using electrophysiological methods. NS11021 at concentrations above 0.3 μM activated KCa1.1 in a concentration-dependent manner by parallel-shifting the channel activation curves to more negative potentials. Single-channel analysis revealed that NS11021 increased the open probability of the channel by altering gating kinetics without affecting the single-channel conductance. NS11021 (10 μM) influenced neither a number of cloned Kv channels nor endogenous Na+ and Ca2+ channels (L- and T-type) in guinea pig cardiac myocytes. In conclusion, NS11021 is a novel KCa1.1 channel activator with better specificity and a 10 times higher potency compared with the most broadly applied KCa1.1 opener, NS1619. Thus, NS11021 might be a valuable tool compound when addressing the physiological and pathophysiological roles of KCa1.1 channels.

The acrylamide (S)-1 differentially affects Kv7 (KCNQ) potassium channels

The family of Kv7 (KCNQ) potassium channels consists of five members. Kv7.2 and 3 are the primary molecular correlates of the M-current, but also Kv7.4 and Kv7.5 display M-current characteristics. M-channel modulators include blockers (e.g., linopirdine) for cognition enhancement and openers (e.g., retigabine) for treatment of epilepsy and neuropathic pain. We investigated the effect of a Bristol-Myers Squibb compound (S)-N-[1-(3-morpholin-4-yl-phenyl)-ethyl]-3-phenyl-acrylamide [(S)-1] on cloned human Kv7.1-5 potassium channels expressed in Xenopus laevis oocytes. Using two-electrode voltage-clamp recordings we found that (S)-1 blocks Kv7.1 and Kv7.1/KCNE1 currents. In contrast, (S)-1 produced a hyperpolarizing shift of the activation curve for Kv7.2, Kv7.2/Kv7.3, Kv7.4 and Kv7.5. Further, the compound enhanced the maximal current amplitude at all potentials for Kv7.4 and Kv7.5 whereas the combined activation/block of Kv7.2 and Kv7.2/3 was strongly voltage-dependent. The tryptophan residue 242 in S5, known to be crucial for the effect of retigabine, was also shown to be critical for the enhancing effect of (S)-1 and BMS204352. Furthermore, no additive effect on Kv7.4 current amplitude was observed when both retigabine and (S)-1 or BMS204352 were applied simultaneously. In conclusion, (S)-1 differentially affects the Kv7 channel subtypes and is dependent on a single tryptophan for the current enhancing effect in Kv7.4.

Managing the complexity of communication: regulation of gap junctions by post-translational modification

Gap junctions are comprised of connexins that form cell-to-cell channels which couple neighboring cells to accommodate the exchange of information. The need for communication does, however, change over time and therefore must be tightly controlled. Although the regulation of connexin protein expression by transcription and translation is of great importance, the trafficking, channel activity and degradation are also under tight control. The function of connexins can be regulated by several post translational modifications, which affect numerous parameters; including number of channels, open probability, single channel conductance or selectivity. The most extensively investigated post translational modifications are phosphorylations, which have been documented in all mammalian connexins. Besides phosphorylations, some connexins are known to be ubiquitinated, SUMOylated, nitrosylated, hydroxylated, acetylated, methylated, and γ-carboxyglutamated. The aim of the present review is to summarize our current knowledge of post translational regulation of the connexin family of proteins.

A Novel KCND3 Gain-of-Function Mutation Associated with Early-Onset of Persistent Lone Atrial Fibrillation

AIMS Atrial fibrillation (AF) is the most common cardiac arrhythmia, and early-onset lone AF has been linked to mutations in genes encoding ion channels. Mutations in the pore forming subunit KV4.3 leading to an increase in the transient outward potassium current (Ito) have previously been associated with the Brugada Syndrome. Here we aim to determine if mutations in KV4.3 or in the auxiliary subunit K(+) Channel-Interacting Protein (KChIP) 2 are associated with early-onset lone AF. METHODS AND RESULTS Two hundred and nine unrelated early-onset lone AF patients (<40 years) were recruited. The entire coding sequence of KCND3 and KCNIP2 was bidirectionally sequenced. One novel non-synonymous mutation A545P was found in KCND3 and was neither present in the control group (n = 432 alleles) nor in any publicly available database. The proband had onset of persistent AF at the age of 22, and no mutations in genes previously associated with AF were found. Electrophysiological analysis of KV4.3-A545P expressed in CHO-K1 cells, revealed that peak-current density was increased and the onset of inactivation was slower compared with WT, resulting in a significant gain-of-function both in the absence and the presence of KChIP2. CONCLUSION Gain-of-function mutations in KV4.3 have previously been described in Brugada Syndrome, however, this is the first report of a KV4.3 gain-of-function mutation in early-onset lone AF. This association of KV4.3 gain-of-function and early-onset lone AF further supports the hypothesis that increased potassium current enhances AF susceptibility.

A Novel Nonsense Variant in Nav1.5 Cofactor MOG1 Eliminates Its Sodium Current Increasing Effect and May Increase the Risk of Arrhythmias

BACKGROUND The protein MOG1 is a cofactor of the cardiac sodium channel, Nav1.5. Overexpression of MOG1 in Nav1.5-expressing cells increases sodium current markedly. Mutations in the genes encoding Nav1.5 and its accessory proteins have been associated with cardiac arrhythmias of significant clinical impact. We sought to investigate whether MOG1 is implicated in cardiac arrhythmias. METHODS We performed a genetic screening of the MOG1-encoding gene (gene symbol RANGRF, alias MOG1) in 220 Danish patients with cardiac arrhythmia. Of the 220, 197 were young patients with lone atrial fibrillation and 23 were patients with Brugada syndrome. The effect of one variant was investigated functionally by patch-clamping CHO-K1 cells coexpressing Nav1.5 with MOG1. RESULTS We uncovered a novel heterozygous nonsense variant, c.181G>T (p.E61X), that, however, was also present in control subjects, albeit at a lower frequency (1.8% vs 0.4%, P = 0.078). Electrophysiological investigation showed that the p.E61X variant completely eliminates the sodium current-increasing effect of MOG1 and thereby causes loss of function in the sodium current. When mimicking heterozygosity by coexpression of Nav1.5 with wild-type MOG1 and p.E61X-MOG1, no current decrease was seen. CONCLUSIONS Our screening of Nav1.5 cofactor MOG1 uncovered a novel nonsense variant that appeared to be present at a higher frequency among patients than control subjects. This variant causes MOG1 loss of function and therefore might be disease causing or modifying under certain conditions.

A Common Single Nucleotide Polymorphism Can Exacerbate Long-QT Type 2 Syndrome Leading to Sudden Infant Death

Background—Identification of infants at risk for sudden arrhythmic death remains one of the leading challenges of modern medicine. We present a family in which a common polymorphism (single nucleotide polymorphism) inherited from the father, combined with a stop codon mutation inherited from the mother (both asymptomatic), led to 2 cases of sudden infant death. Methods and Results—KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, CACNA1c, CACNB2b, and KCNJ2 genes were amplified and analyzed by direct sequencing. Functional electrophysiological studies were performed with the single nucleotide polymorphism and mutation expressed singly and in combination in Chinese ovary (CHO-K1) and COS-1 cells. An asymptomatic woman presenting after the death of her 2-day-old infant and spontaneous abortion of a second baby in the first trimester was referred for genetic analysis. The newborn infant had nearly incessant ventricular tachycardia while in utero and a prolonged QTc (560 ms). The mother was asymptomatic but displayed a prolonged QTc. Genetic screening of the mother revealed a heterozygous nonsense mutation (P926AfsX14) in KCNH2, predicting a stop codon. The father was asymptomatic with a normal QTc but had a heterozygous polymorphism (K897T) in KCNH2. The baby who died at 2 days of age and the aborted fetus inherited both K897T and P926AfsX14. Heterologous coexpression of K897T and P926AfsX14 led to loss of function of HERG current much greater than expression of K897T or P926AfsX14 alone. Conclusions—Our data suggest that a common polymorphism (K897T) can markedly accentuate the loss of function of mildly defective HERG channels, leading to long-QT syndrome–mediated arrhythmias and sudden infant death.

Differential effects of the transient outward K+ current activator NS5806 in the canine left ventricle

To examine the electrophysiological and molecular properties of the transient outward current (I(to)) in canine left ventricle using a novel I(to) activator, NS5806, I(to) was measured in isolated epicardial (Epi), midmyocardial (Mid) and endocardial (Endo) cells using whole-cell patch-clamp techniques. NS5806 activation of K(v)4.3 current was also studied in CHO-K1 cells and Xenopus laevis oocytes. In CHO-K1 cells co-transfected with K(v)4.3 and KChIP2, NS5806 (10 microM) caused a 35% increase in current amplitude and a marked slowing of current decay with tau increasing from 7.0+/-0.4 to 10.2+/-0.3 ms. In the absence of KChIP2, current decay was unaffected by NS5806. In ventricular myocytes, NS5806 increased I(to) density by 80%, 82%, and 16% in Epi, Mid, and Endo myocytes, respectively (at +40 mV) and shifted steady-state inactivation to negative potentials. NS5806 also significantly slowed decay of I(to), increasing total charge to 227%, 192% and 83% of control in Epi, Mid and Endo cells, respectively (+40 mV, p<0.05). Quantification of K(v)4.3 and KChIP2 mRNA in the 3 ventricular cell types revealed that levels of K(v)4.3 message was uniform but those of KChIP2 were significantly greater in Epi and Mid cells. The KChIP2 gradient was confirmed at the protein level by Western blot. Our results suggest that NS5806 augments I(to) by increasing current density and slowing decay and that both depend on the presence of KChIP2. I(to) and its augmentation by NS5806 are greatest in Epi and Mid cells because KChIP2 levels are highest in these cell types.

Effect of the Ito activator NS5806 on cloned Kv4 channels depends on the accessory protein KChIP2

The compound NS5806 increases the transient outward current (Ito) in canine ventricular cardiomyocytes and slows current decay. In human and canine ventricle, Ito is thought to be mediated by KV4.3 and various ancillary proteins, yet, the exact subunit composition of Ito channels is still debated. Here we characterize the effect of NS5806 on heterologously expressed putative Ito channel subunits and other potassium channels.

Physiological consequences of transient outward K+ current activation during heart failure in the canine left ventricle

BACKGROUND Remodeling of ion channel expression is well established in heart failure (HF). We determined the extent to which I(to) is reduced in tachypacing-induced HF and assessed the ability of an I(to) activator (NS5806) to recover this current. METHOD AND RESULTS Whole-cell patch clamp was used to record I(to) in epicardial (Epi) ventricular myocytes. Epi- and endocardial action potentials were recorded from left ventricular wedge preparations. Right ventricular tachypacing-induced heart failure reduced I(to) density in Epi myocytes (Control=22.1±1.9pA/pF vs 16.1±1.4 after 2weeks and 10.7±1.4pA/pF after 5 weeks, +50mV). Current decay as well as recovery of I(to) from inactivation progressively slowed with the development of heart failure. Reduction of I(to) density was paralleled by a reduction in phase 1 magnitude, epicardial action potential notch and J wave amplitude recorded from coronary-perfused left ventricular wedge preparations. NS5806 increased I(to) (at +50mV) from 16.1±1.4 to 23.9±2.1pA/pF (p<0.05) at 2weeks and from 10.7±1.4 to 14.4±1.9pA/pF (p<0.05) in 5 weeks tachypaced dogs. NS5806 increased both fast and slow phases of I(to) recovery in 2 and 5-week HF cells and restored the action potential notch and J wave in wedge preparations from HF dogs. CONCLUSIONS The I(to) agonist NS5806 increases the rate of recovery and density of I(to), thus reversing the HF-induced reduction in these parameters. In wedge preparations from HF dogs, NS5806 restored the spike-and-dome morphology of the Epi action potential providing proof of principal that some aspects of electrical remodelling during HF can be pharmacologically reversed.