Control of Rhythm and Rate278Cx43 hemichannels in ventricular cardiomyocytes can be activated by an elevation of cytoplasmic Ca2+ through a CaM-dependent signaling cascade and are a potent contributor to cardiac arrhythmogenesis279Exploration of the dominant-negative effect of a SCN5A mutation in mice using adeno-associated viruses280Modeling susceptibility to drug-induced long qt syndrome with a panel of subject-specific induced pluripotent stem cells

# 278 Cx43 hemichannels in ventricular cardiomyocytes can be activated by an elevation of cytoplasmic Ca2+ through a CaM-dependent signaling cascade and are a potent contributor to cardiac arrhythmogenesis {#article-title-2}Introduction and purpose: Cx43, most abundantly expressed in the ventricles, is engaged in forming both gap junctions that electrically couple the cardiac tissue and unapposed hemichannels (HCs) that function as a non-selective transmembrane conduit. The latter are typically closed but open in response to electrical or chemical triggers such as pro-inflammatory cytokines or elevation of cytoplasmic Ca2+ ([Ca2+]i). Evidence for hemichannel opening by [Ca2+]i elevation is based on indirect ATP/dye uptake measurements, but conclusive electrophysiological evidence is lacking. Here, we studied [Ca2+]i-dependent activation of cardiac Cx43 HCs with the use of single-channel recordings and further proposed their arrhythmogenic potential.Methods: Left ventricular cardiomyocytes were acutely isolated from C57/Bl6 and inducible Cx43 knockdown mice (Cx43Cre-ER(T)/Fl). We provoked Ca2+ release from the sarcoplasmic reticulum by local application of caffeine (10 mM) under voltage-clamp conditions (Vm = -70 mV) and studied the effect on HC unitary currents using whole-cell patch-clamp techniques. Involvement of Cx43 HCs was verified by the biophysical properties, intracellular application of Gap19 (100 µM) and CT9 (100 µM) (a selective Cx43 HC blocker and positive modulator respectively) and Cx43 knockdown. Involvement of [Ca2+]i was assessed by applying the Ca2+-chelator EGTA (10 mM) intracellularly. Calmodulin (CaM) antagonist W7 (50 µM) was used to investigate the contribution of CaM signaling.Results and conclusions: Applying caffeine pulses (8 s) at resting membrane potential activated an inward current carried by the Na+/Ca2+ exchanger (NCX). Superimposed on this macroscopic current, there appeared microscopic current spiking activity characterized by a unitary conductance of ~210 pS. Unitary currents were inhibited by EGTA, by conditional Cx43 knockdown (Cx43Cre-ER(T)/Fl) and by the Cx43 HC blocking peptide Gap19. Additionally, CT9 peptide promoted the HC currents activated by caffeine-induced Ca2+-release while delivery of W7 into the cell completely abolished HC currents. The results indicate that Cx43 HCs can be opened by Ca2+-stimulation without any associated electrical stimulus through a CaM-dependent signaling cascade. The kinetics of Ca2+-triggered opening activity were very different from voltage-triggered activity (fast spiking versus prolonged opening respectively) indicating a distinct gating mechanism. Computational modeling of the impact of electrically/chemically-triggered Cx43 HC opening on cardiomyocyte excitability indicates that this may contribute to arrhythmogenesis in the heart by providing depolarizing inward currents with potential for generating delayed afterdepolarizations and subsequent focal excitation.# 279 Exploration of the dominant-negative effect of a SCN5A mutation in mice using adeno-associated viruses {#article-title-3}Introduction: Loss-of-function mutations in the cardiac sodium (Na+) channel α-subunit gene, SCN5A, cause Brugada syndrome (BrS), a hereditary disease with an autosomal dominant transmission and variable penetrance, characterized by ventricular fibrillation and sudden cardiac death. We previously evidenced in HEK293 cells, the dominant-negative effect of the R104W BrS mutation in the N-terminal region of the cardiac Na+ channel, inducing the retention of the wild-type (WT) channel and the proteasomal degradation of the mutant protein, leading to a drastic reduction of the resulting Na+ current. Our goal was to further study the effects of this mutation in vivo in mice, taking advantage of adeno-associated viruses (AAV) and pre-mRNA trans-splicing strategy.Methods: Because of the large size of the SCN5A gene, we used a dual AAV vector strategy combining viral DNA recombination and trans-splicing. Mice were injected with two AAV-9: one, AAV-SCN5A-5u0027, packaging the cardiac troponin-T promoter, the 5u0027 half of hSCN5A, the 5u0027 donor site of a synthetic intron and a highly recombinogenic part of the human alkaline phosphatase (AP); and another, AAV-SCN5A-3u0027, packaging the same AP sequence, the 3u0027 acceptor site of the intron, the 3u0027 half of hSCN5A fused to the gfp gene as a reporter and the SV40 polyA signal. Six weeks after systemic injection, the hSCN5A-GFP full-gene expression was assessed by RT-qPCR, western blot and immunohistochemistry on transduced heart tissues. The Na+ current was recorded by the patch-clamp technique in isolated adult trasduced cardiomyocytes.Results: Almost 40% of cells were transduced in injected mice hearts. Overexpression of the R104W mutant channel induced a dilatation of the left vendtricle (+21% in systole, Pu003c0.001), a reduction of the density of the full-activated Na+ current (-14% at -30mV, Pu003c0.05) and a rightward shift of the activation curve of the Na+ current (+3mV, Pu003c0.005), compared to control mice. In contrast, overexpression of the WT human Na+ channel led to a significant reduction of the PR interval on the ECG (-30%, Pu003c0.001), and to a significant increase in the Na+ current (+44% at -30mV, Pu003c0.001) without changes in the activation parameters compared to non-injected mice.Conclusion: Altogether, our data suggested that the trans-splicing and viral DNA recombination are a successful strategy to overexpress the Na+ channel in mouse hearts. By this approach, we reproduced the dominant-negative effects of the R104W mutation, previously observed in vitro, on the endogenous cardiac Na+ current in adult mice.# 280 Modeling susceptibility to drug-induced long qt syndrome with a panel of subject-specific induced pluripotent stem cells {#article-title-4}Introduction: A high number of drugs can induce prolongation of cardiac repolarization and trigger life-threatening cardiac arrhythmias. However, current methods to predict drug-induced QT prolongation (diLQT) are suboptimal. In addition, the risk of developing diLQT varies markedly between subjects. Multiple mutations in genes encoding ion channels have been reported to underlie the congenital form of the long QT syndrome (LQTS). However, most people developing diLQTS do not have mutations in LQTS genes and the risk of developing diLQT is more likely associated with a predisposing genetic background with multiple genetic factors affecting the cardiac repolarization reserve.Purpose: We investigated whether induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) from subjects with quantitative measure of diLQT in vivo can recapitulate the phenotype in vitro thereby providing a model system for diLQT.Methods: We evaluated cardiac repolarization of 92 healthy subjects in response to a pharmacological challenge with a single oral dose (80mg) of Sotalol. We collected skin fibroblasts from twenty subjects with the most extreme responses to Sotalol (10 subjects with high-sensitivity (high-S) and 10 with low-sensitivity (low-S); change in QT interval corrected according to Fridericiau0027s formula, QTcf: 48.5±7.1ms vs. 0.2±4.8ms). Field potential duration (FPD), an analogous to the QT interval in the electrocardiogram (ECG), was measured in iPSC-CMs by multi-electrode array (MEA) under basal conditions and in response to increasing concentrations of Sotalol.Results: Cardiac differentiation resulted in the generation of iPSC-CMs with appropriate cardiac channel expression and response to a hERG blocker E4031, in 15 lines (8 vs. 7 lines in low-S and high-S groups respectively). MEA recordings showed a significantly higher response to Sotalol in iPSC-CMs from high-S compared to low-S subjects (maximal response to Sotalol: 52±6% vs. 29±6%, pu003c0.02). Five of the 7 lines derived from the high-S group displayed arrhythmias in response to sotalol stimulation while arrhythmias only occurred in one line from the low-S group. Transcriptomic profiling identified dysregulation of genes (DLG2, KCNE4, PTRF, HTR2C, CAMKV) involved in downstream regulation of cardiac repolarization machinery as underlying high sensitivity to Sotalol.Conclusions: Our findings suggest that patient-specific iPSCs can be used to model the functional abnormalities observed in diLQTS and offer novel insights for the development of iPSC-based screening assays for toxic drug reactions.