Table1_Electrophysiological basis of cardiac arrhythmia in a mouse model of myotonic dystrophy type 1.DOCX

Introduction: Myotonic dystrophy type 1 (DM1) is a multisystemic genetic disorder caused by the increased number of CTG repeats in 3′ UTR of Dystrophia Myotonia Protein Kinase (DMPK) gene. DM1 patients experience conduction abnormalities as well as atrial and ventricular arrhythmias with increased susceptibility to sudden cardiac death. The ionic basis of these electrical abnormalities is poorly understood.Methods: We evaluated the surface electrocardiogram (ECG) and key ion currents underlying the action potential (AP) in a mouse model of DM1, DMSXL, which express over 1000 CTG repeats. Sodium current (INa), L-type calcium current (ICaL), transient outward potassium current (Ito), and APs were recorded using the patch-clamp technique.Results: Arrhythmic events on the ECG including sinus bradycardia, conduction defects, and premature ventricular and atrial arrhythmias were observed in DMSXL homozygous mice but not in WT mice. PR interval shortening was observed in homozygous mice while ECG parameters such as QRS duration, and QTc did not change. Further, flecainide prolonged PR, QRS, and QTc visually in DMSXL homozygous mice. At the single ventricular myocyte level, we observed a reduced current density for Ito and ICaL with a positive shift in steady state activation of L-type calcium channels carrying ICaL in DMSXL homozygous mice compared with WT mice. INa densities and action potential duration did not change between DMSXL and WT mice.Conclusion: The reduced current densities of Ito, and ICaL and alterations in gating properties in L-type calcium channels may contribute to the ECG abnormalities in the DMSXL mouse model of DM1. These findings open new avenues for novel targeted therapeutics.</p

Proton current-voltage relationship of the Na_v1.5/R219H channel recorded in an NMDG Na⁺-free solution.

<p>(<b>A</b>) Representative proton current traces from oocytes expressing the Na_v1.5/R219H channel recorded at pH_o 8.40, 7.40, 6.80, and 6.00, as indicated, in response to 200 ms voltage steps ranging from −140 mV to +40 mV in 5-mV increments from a holding potential of −80 mV (the protocol is given in the centre inset), without on-line leak subtraction. The dashed line represents the zero current. For clarity, only current every 10 mV are shown. (<b>B</b>) Current-voltage relationship where the currents in (<b>A</b>) were plotted as a function of the test potential (5 mV increments), after offline linear leak subtraction. Reversal potential determined in a Na⁺-free NMDG solution at pH_o 8.40 using voltage steps as described in (<b>A</b>). The pH_i was measured using a pH-sensitive electrode. Similar results were obtained with four separate batches of oocytes. The inset shows the pH_oand pH_i values and between parentheses is the predicted values calculated using the Nernst equation. The bleu trace shows the voltage-dependent of activation (Q–V), the grey zone illustrates the transitional zone corresponding to the probability of the voltage sensor being stabilized in the outward position. (<b>C</b>) Correlation between the peak Na⁺ current measured in Ringer's solution and the proton current measured at −140 mV and pH_o 4.00 (n = 31) on the same oocytes. The data were obtained from one batch of oocytes over three days. The straight line represents the linear regression of the data set and R² is the correlation coefficient and shows the goodness of fit. Similar results were obtained with three separate batches of oocytes. (<b>D</b>) Proton currents measured in response to a change in pH_o at −140 mV in an NMDG Na⁺-free solution. The currents were normalized to the currents obtained at pH_o = 4.00 for each cell. The mean data (n = 5) was fitted to the Henderson-Hasselbach equation, 1/[1+exp(2.3(pH_o−pK_a))]. Error bars are smaller than the symbols.</p

Family pedigree, clinical evaluation, and molecular genetics.

<p>(<b>A</b>) The index patient (III-1) is indicated by an arrow. Individuals indicated with black squares/circles carry the mutation and a clinical phenotype (III-1, III-2, II-2). Individuals indicated with grey circles (II-3 to II-5) were clinically diagnosed with DCM, but not genotyped. Abbreviation: DCM (dilated cardiomyopathy). (<b>B</b>) 12-lead ECG of the index patient showing third degree AV-block with a ventricular escape rhythm and a small QRS-complex with a heart rate of 43 bpm (artefact in lead V1). (<b>C</b>) Non-sustained ventricular tachycardia (220 bpm) occurred at a heart rate of 130 bpm and a work load of 192 W during an exercise stress test. (<b>D</b>) Different DHPLC eluting profiles at 59.8°C of the PCR products of exon 6 in the index patient compared to the control. Abbreviation: DHPLC (denaturing high performance liquid chromatography). (<b>E</b>) A heterozygous change of arginine CGC (R) to histidine CAC (H) resulted in the missense mutation R219H. (<b>F</b>) Sequence alignments of the S4 of domain 1 from Na⁺ and K⁺ (<i>Shaker</i> B) channels in different species.</p

Na_v1.5/R219H induces an inward proton current and intracellular acidification.

<p><i>Xenopus</i> oocytes expressing Na_v1.5/WT or Na_v1.5/R219H channel were impaled with three electrodes, one filled with an H⁺ resin to measure pH_i, and two to clamp the oocyte at −80 mV in a Na⁺-free NMDG solution containing 1 µM TTX, as indicated. Typical proton current recordings (red traces) in response to different pH_o value and the pH_i measurement rate (bleu traces) from an oocyte expressing the Na_v1.5/R219H (<b>A</b>) or Na_v1.5/WT channel (<b>B</b>). Intracellular pH_i values before changing solutions in experiments similar to (<b>A</b>) and (<b>B</b>) were plotted against pH_o (***, p<0.001 compared to WT, n = 10–19)(<b>C</b>). Similar recordings were obtained with four batches of oocytes. (<b>D</b>) Changes in pH_i after incubating oocytes expressing the Na_v1.5/WT (triangles) or Na_v1.5/R219H (squares) channel, or water-injected oocytes (circles) in OR3 medium at different pH_o values (***, p<0.001, **; p<0.01; *, p<0.05; compared to WT, n = 7–13). pH_i measurements were carried out in Ringer's solution at pH_o of 7.40.</p

Biophysical characterization of the Na_v1.5/R219H DCM mutation proton current recordings.

<p>Representative current traces recorded using the cut-open oocyte technique from Na_v1.5/WT (<b>A</b>) and Na_v1.5/R219H (<b>B</b>) channels. Currents were elicited by depolarizing pulses from −100 mV to +60 mV, with 10 mV increments for each step. (<b>C</b>) The voltage dependence of steady-state activation and inactivation of WT (activation, n = 7; inactivation, n = 8) and R219H (activation, n = 8; inactivation, n = 8). Activation curves were derived from <i>I</i>–<i>V</i> curves and fitted to a standard Boltzmann equation: <i>G</i> (<i>V</i>)/<i>G </i>_max = 1/(1+exp ((<i>V</i>−<i>V</i>_1/2)/<i>k_v</i>)), with midpoints (V_1/2) is slow factors (<i>k_v</i>) listed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038331#pone.0038331.s001" target="_blank">Table S1</a></b>. The voltage-dependence of inactivation was induced by applying conditioning pre-pulses to membrane potentials ranging from a holding potential of −150 to −20 mV for 500 ms with 10 mV increments and was then measured using a 20-ms test pulse to −30 mV for each step (see protocol in inset). The recorded inactivation data were fitted to a standard Boltzmann equation: <i>I</i> (<i>V</i>)/<i>I</i>_max = 1/(1+exp ((<i>V</i>−<i>V</i>_1/2)/<i>k_v</i>)), with midpoints (<i>V</i>_1/2) is slow factors (<i>k_v</i>) listed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038331#pone.0038331.s001" target="_blank">Table S1</a></b>. (<b>D</b>) Time courses of recovery from inactivation of Na_v1.5/WT and Na_v1.5/R219H channels. A 40 ms conditioning pre-pulse was used to monitor recovery using a 20-ms test pulse after a variable recovery interval ranging from 5 to 500 ms (see protocol in inset). A single-exponential function was used to determine the time constants of recovery.</p

Use-dependent sommation of modified channels and tail current inactivation kinetics in ALNs.

<p>Mean time course of pyrethroid-modified channels along with protocol shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112194#pone-0112194-g002" target="_blank">Figure 2</a> (n = 7, 10 and 10 ALNs for cypermethrin, permethrin and tetramethrin, respectively). The percentage of modified channels is calculated from tail currents amplitude according to Eqn 3. Whereas cypermethrin and permethrin induce an increase in % of modified channels, tetramethrin shows the opposite effect. B2. Kinectics of tail currents estimated by the R600 value, i.e. the percentage of residual tail current 600 ms after the end of the tenth pulse of the 10-pulse protocol. Tetramethrin induces faster decaying tail currents than cypermethrin or permethrin.</p

Gating parameters of WT and R1432G Na_v1.5 channels expressed in HEK293T cells.

<p>Abbreviations are: <i>n</i>, number of cells per group; <i>k</i>, slope factor of voltage dependence of (in)activation (mV) and V_1/2, voltage of half-maximal (in)activation (mV). Statistically significant results were determined using one-way analysis of variance (ANOVA) with Bonferroni's post hoc tests. ^*, P<0.05; ^**, P<0.01 (<i>vs</i> WT/(-) + β₁); ^†, P<0.05; ^††, P<0.01 (<i>vs</i> WT/R1432G + β₁).</p

Abolition of R1432G dominant-negative effect in the absence of the β₁-subunit.

<p><b>A.</b> IV curves of Na_v1.5 currents recorded from HEK293T cells transfected with WT and/or R1432G channels without β₁-subunit (n = 9–10). <b>B.</b> Average peak current densities WT and/or R1432G channels at -40 mV. n.s., p>0.05 <i>versus</i> WT/R1432G condition (Student <i>t</i>-test). <b>C.</b> Voltage-dependence of activation (open symbols, n = 9–10) and inactivation (filled symbols, n = 7) of WT and/or R1432G Na_v1.5 expressed in HEK293T without β₁. Inactivation and activation data were obtained using standard pulse protocols shown in inset and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048690#pone-0048690-g001" target="_blank">Figure 1A</a> respectively. (In)activation parameters are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048690#pone-0048690-t001" target="_blank">Table 1</a>. <b>D.</b> Quantification by luminometric ELISA assay of FLAG-tagged Na_v1.5/WT channels alone (white bars, n = 6) or with R1432G (grey bars, n = 6) in the absence of β₁-subunit. The bar chart shows surface and total amount of WT-FLAG channels from nonpermeabilized cells (solid bars) and permeabilized cells (hatched bars) respectively in the absence of β₁-subunit. Data are presented as relative light unit (RLU) normalized to the total WT-FLAG/(-) condition and are expressed as mean ± SEM. n.s. indicates no significant difference with surface WT-FLAG/(-) condition (<i>t</i>-test).</p

Dose-response curve of permethrin-modified sodium channels in ALNs.

<p>Percentage of permethrin-modified channels, calculated according to Eqn 3, from the permethrin-induced tail current amplitude measured 3ms after a single pulse (of 3 ms in duration, see inset). The percentage of modified channels increases as a function of permethrin concentration (filled circle, n = 7, 10, 25 and 9 at 0.1, 1, 10 and 50 µM respectively). The same curve but recorded in ORNs (from Kadala et al. 2011) is shown for comparison (empty circles, n = 3, 9, 14, 5).</p

R1432G mutant alters WT channel localization to the cell membrane in the presence of β₁-subunit.

<p><b>A.</b> Confocal imaging of Na channels in cells co-expressing R1432G mutant and Na_v1.5/WT-FLAG or expressing Na_v1.5/WT-FLAG alone. Immunocytochemistry with anti-FLAG antibodies was performed first on fixed non-permeabilized cells to detect surface FLAG-tagged WT channels (green, left panels). The same cells were then permeabilized to label total mutant and WT Na_v1.5 proteins with anti-Na_v1.5 antibodies (red, middle panels). Light transmission imaging of the HEK293T cells are presented on the right panels. Scale bar, 10 µm. <b>B.</b> Quantification by luminometry of FLAG-tagged Na_v1.5/WT channels in the presence (gray bars) or the absence (white bars) of R1432G subunits in HEK293T cells. Surface and total amount of WT-FLAG channels was measured from non-permeabilized (solid bars) and permeabilized (hatched bars) cells respectively. Data are presented as relative light unit (RLU) normalized to the total WT-FLAG/(-) condition. Data are expressed as mean ± SEM of six independent experiments. *, P<0.05, indicates significant difference with surface WT-FLAG/( ) condition (<i>t</i>-test). All experiments were realized in the presence of the β₁-subunit.</p