DataSheet1.pdf

<p>In cardiomyocytes, the voltage-gated transient outward potassium current (I_to) is responsible for the phase-1 repolarization of the action potential (AP). Gain-of-function mutations in KCND3, the gene encoding the I_to carrying K_V4.3 channel, have been associated with Brugada syndrome (BrS). While the role of I_to in the pro-arrhythmic mechanism of BrS has been debated, recent studies have suggested that an increased I_to may directly affect cardiac conduction. However, the effects of an increased I_to on AP upstroke velocity or sodium current at the cellular level remain unknown. We here investigated the consequences of K_V4.3 overexpression on Na_V1.5 current and consequent sodium channel availability. We found that overexpression of K_V4.3 protein in HEK293 cells stably expressing Na_V1.5 (HEK293-Na_V1.5 cells) significantly reduced Na_V1.5 current density without affecting its kinetic properties. In addition, K_V4.3 overexpression decreased AP upstroke velocity in HEK293-Na_V1.5 cells, as measured with the alternating voltage/current clamp technique. These effects of K_V4.3 could not be explained by alterations in total Na_V1.5 protein expression. Using computer simulations employing a multicellular in silico model, we furthermore demonstrate that the experimentally observed increase in K_V4.3 current and concurrent decrease in Na_V1.5 current may result in a loss of conduction, underlining the potential functional relevance of our findings. This study gives the first proof of concept that K_V4.3 directly impacts on Na_V1.5 current. Future studies employing appropriate disease models should explore the potential electrophysiological implications in (patho)physiological conditions, including BrS associated with KCND3 gain-of-function mutations.</p

1493delK mutant and wild-type (WT) human cardiac sodium channel current expressed in HEK293 cells.

<p>(A) Whole-cell sodium current traces in response to increasing step depolarizations in WT (left) and 1493delK (right). (B) Voltage protocols for activation and steady-state inactivation. (C) Averaged sodium current– voltage relation for WT and 1493delK sodium channels. (D) Bar histogram showing averaged WT and 1493delK sodium peak currents at −20 mV. (E) Average voltage-dependence of activation and steady-state inactivation for wild-type (WT) and 1493delK sodium channels. For the activation curve, normalized peak conductance was plotted as a function of the membrane potential. For the inactivation curve, peak sodium currents were normalized to maximum values in each cell and plotted as a function of the voltage of the conditioning step.</p

Clinical and Genetic Characteristics of <i>SCN5A</i> 1493delK Carriers.

<p>ECG, electrocardiogram; ICD, implantable cardioverter defibrillator; PM, Pacemaker; m, male; f, female; y, years; +, mutation carrier or ICD implanted; (+),obligate mutation carrier; SCD, sudden cardiac death; RV, right ventricle; CAD, coronary artery disease; #, by transthoracic echocardiographic; §, by magnetic resonance imaging; $, by ventriculography; n.p., not performed; n.a., not avalible; PWD, P-wave duration; AVB I°, atrioventricular block first-degree; CCD, cardiac conduction delay; RFB, right fascicular block; LAFB, left anterior fascicular block. Propositus (10021_149) printed in italics.</p

Inactivation kinetics of 1493delK mutant and wild-type (WT) human cardiac sodium channels.

<p>(A) Time course of current decay. (A-i) Fast and slow time constants of current decay for WT and 1493delK sodium channels are plotted as a function of membrane potential. Asterisks indicate statistical significance (p<0.05). (A-ii) Ratio of the amplitudes of fast and slow inactivation time constants plotted as a function of voltage for WT and 1493delK sodium channels. (B) Time course of recovery from inactivation for WT and 1493delK sodium channels. Peak sodium currents elicited by P2 were normalized (P2/P1) and plotted as a function of the recovery interval. Inset: 2-pulse protocol. (C) Development of slow inactivation for WT and 1493delK sodium channels. Peak sodium currents elicited by P2 were normalized (P2/P1) and plotted as a function of the duration of the conditioning step (P1). Inset: 2-pulse protocol.</p

Electrophysiological characteristics of WT and 1493delK mutant sodium channels in HEK293 cells.

<p>V_1/2, voltage of half-maximal (in)activation; k, slope factor of voltage- dependence of (in)activation; A, fraction of channels that enter the slow inactivated state at t = 1 s; <i>τ</i>, time constant for development of slow inactivation; <i>τ</i>_f, fast time constant of recovery from inactivation; <i>τ</i>_s, slow time constant of recovery from inactivation.</p>*<p>p<0.05 vs WT (Student’s t-test).</p

Electrocardiogram Parameters of Mutation or Obligate Mutation Carriers.

<p>HR, heart rate; P, P interval; PQ, PQ interval; QRS, QRS interval; QTc, corrected QT interval; m, male; f, female; SD, standard deviation.</p

Topological model of the cardiac sodium channel (Na_V1.5).

<p>Location of the mutations in the linker region between domains DIII and DIV that is responsible for the inactivation of the channel.</p

Sodium channel membrane expression in wild-type and mutant 1493delK <i>SCN5A</i>-transfected HEK293 cells.

<p>Confocal immunofluorescence of the a-subunit of cardiac sodium channel (Na_V1.5) and the endoplasmic reticulum transmembrane protein calnexin in HEK293 expressing WT (left) and mutant 1493delK (right) sodium channels. Top and middle panels show staining with anti-Na_V1.5 (green) and anti-calnexin (red) respectively. Bottom panels show overlay of red and green channels of double staining with anti-Na_V1.5 (green) and anti-calnexin (red) antibodies. Membrane labeling for Na_V1.5 is observed as a clearly distinguishable green rim surrounding the intracellularly located calnexin (red) in WT <i>SCN5A</i> transfected HEK293 cells, whereas mutant 1493delK <i>SCN5A</i> transfected HEK293 cells do not show clear cell-surface labeling, but mostly cytoplasmic Na_V1.5 staining. Scale bars indicate 25 µm.</p

Genotype effects at rs13477506.

<p>(A) <i>Tnni3k</i> expression (Illumina probe ILMN_3023962) as a function of genotype at rs13477506 in F2 mice; homozygous 129P2: AA, green; 129P2-FVBN/J heterozygous: AB, turquoise; homozygous FVBN/J: BB, blue; the darker shades represent the independent validation of the <i>Tnni3k</i> transcription levels by Q-PCR (right y-axis). (B) PR interval as a function of genotype at rs13477506 in F2 mice. Error bars indicate standard errors.</p

Overview of the locations of the linkage regions on chromosome 3.

<p>(A) Entire mouse chromosome 3 with refseq genes indicated in dark blue, black bar: 1.5 LOD drop of the PR-QTL; dark red bar 20 Mb DBA.AKR congenic region; purple bar 1.5 LOD drop of the Tnni3k eQTL; grey bar minimal region of overlap. (B) 1 Mb close up of the minimal region of overlap; the positions of rs49812611 (associated with nonsense mediated decay in DBA/2J) and rs13477506 (QTLs) are indicated. (C) Haplotypes of a panel of 9 inbred mouse strains as determined by the mouse phylogeny viewer (<a href="http://msub.csbio.unc.edu/" target="_blank">http://msub.csbio.unc.edu/</a>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003113#pgen.1003113-Yang1" target="_blank">[19]</a>.</p