0235 : In vivo overexpression of a cardiac sodium channel mutant in mice

Loss-of-function mutations in the cardiac Na+ channel α-subunit gene, SCN5A, cause Brugada syndrome (BrS), a hereditary disease characterized by ventricular fibrillation and sudden cardiac death. We previously evidenced, in HEK cells, the dominant-negative effect of the R104W BrS mutation in Nav1.5, inducing the retention of the wild-type (WT) channel and the proteasomal degradation of the mutant protein. To explore this dominant-negative effect in vivo, we created a murine model using adeno-associated viruses (AAV).We used a dual AAV vector strategy combining viral DNA recombination and trans-splicing. One-week old mice were injected with two AAV serotypes capsid 9: one, packaging the cardiac specific troponin-T promoter, the 5’ half of hSCN5A, the 5’ donor site of a synthetic intron and a highly recombinogenic sequence; and another, packaging the same recombinogenic sequence, the 3’ acceptor site of the synthetic intron, the 3’ half of hSCN5A, the gfp gene as a reporter, and the SV40 polyA signal. Six weeks after injection, the hSCN5A full-gene expression and the percentage of transduced cardiac cells were assessed by qPCR, western blot (WB) analysis and immunohistochemistry on transduced heart tissues. The Na+ current was recorded by the patchclamp technique in isolated cardiomyocytes.Both WT and mutant human Nav1.5 transcripts and proteins were observed by RT-qPCR, WB and immunohistochemistry on injected-mice heart tissues. Patch-clamp recordings in WT-channel injected mice evidenced a two-fold increase of the Na+ current. In contrast, the cardiac Na+ current of R104Winjected mice was impaired (i.e. the current density was decreased by 45% and the activation was shifted by -4mV).Our data suggest that the trans-splicing and viral DNA recombination strategy using AAV9 serotype and a cardiac-specific promoter is successful to overexpress WT or mutant Na+ channels in mouse hearts. This approach allowed us to modulate the cardiac Na+ current in adult mice

Rescue of fragile X syndrome phenotypes in Fmr1 KO mice by a BKCa channel opener molecule.

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Characterization of the automatic catecholaminergic activity in the pulmonary vein of the rat : involvement of the alpha 1 Adrenoceptors

Les mécanismes impliqués dans l’activation de foyers ectopiques dans les veines thoraciques, source de fibrillation auriculaire (FA) chez l’Homme, sont encore méconnus. Nous avons montré que la noradrénaline peut induire une activité automatique sur la veine pulmonaire (VP) mais pas sur l’oreillette gauche (OG) chez le Rat. Au cours de cette étude, nous avons montré : 1) un potentiel de membrane de repos dépolarisé dans la VP et la veine cave supérieure (VCS) par rapport à l’OG ainsi que la présence d’une activité automatique en salves sur la VP ; 2) une dépolarisation due à la stimulation des récepteurs α1-adrénergiques (α1-AR) plus importante sur la VP que sur les deux autres tissus qui induit une inexcitabilité de la VP et la VCS ; 3) la présence d’une activité déclenchée favorisée par la stimulation des β1-AR sur la VCS ; 4) une diminution liée à l’âge de l’incidence de l’activité automatique catécholaminergique et des réponses à la stimulation des α1-AR sur la VP ainsi que la présence d’une activité déclenchée sous stimulation des α1-AR sur la VP chez les animaux âgés. Nous avons donc pu mettre en évidence des différences fonctionnelles entre le myocarde des veines thoraciques et le tissu atrial. Cependant, la relation entre l’activité automatique observée sur la VP du Rat et la FA reste encore à établir.The mechanisms involved in the activation of ectopic foci within the thoracic veins, cause of atrial fibrillation (AF) in man, are still unknown. We shown that norepinephrine (NE) can induce an automatic activity in pulmonary vein (PV) but not in left atrium (LA) of the Rat. In this study, we shown: 1) a depolarised resting membrane potential in PV and superior vena cava (SVC) compared with LA and the occurence of an automatic activity in repetitive bursts in PV; 2) a depolarisation due to α1-adrenergic receptors (α1-AR) stimulation more pronounced in PV than the other two tissues, which induce an inexcitability of the PV and the SVC; 3) the occurence of a triggered activity facilitated by the simulation of β1-AR in SVC; 4) a decrease with age of the incidence of the catecholaminergic automatic activity and responses to the stimulation of α1-AR in PV and the occurence of a triggered activity under stimulation of α1-adrenergic in PV of old animals. Therefore, we have shown some functionnal differences between the myocardium of thoracic veins and atrial tissu. Nevertheless, the relationship between automatic activity observed in Rat PV and AF remains to establish

Caractérisation de l'activité automatique catécholaminergique au niveau de la veine pulmonaire du rat (rôle des récepteurs Alpha 1 Adrénergiques)

Les mécanismes impliqués dans l activation de foyers ectopiques dans les veines thoraciques, source de fibrillation auriculaire (FA) chez l Homme, sont encore méconnus. Nous avons montré que la noradrénaline peut induire une activité automatique sur la veine pulmonaire (VP) mais pas sur l oreillette gauche (OG) chez le Rat. Au cours de cette étude, nous avons montré : 1) un potentiel de membrane de repos dépolarisé dans la VP et la veine cave supérieure (VCS) par rapport à l OG ainsi que la présence d une activité automatique en salves sur la VP ; 2) une dépolarisation due à la stimulation des récepteurs a1-adrénergiques (a1-AR) plus importante sur la VP que sur les deux autres tissus qui induit une inexcitabilité de la VP et la VCS ; 3) la présence d une activité déclenchée favorisée par la stimulation des b1-AR sur la VCS ; 4) une diminution liée à l âge de l incidence de l activité automatique catécholaminergique et des réponses à la stimulation des a1-AR sur la VP ainsi que la présence d une activité déclenchée sous stimulation des a1-AR sur la VP chez les animaux âgés. Nous avons donc pu mettre en évidence des différences fonctionnelles entre le myocarde des veines thoraciques et le tissu atrial. Cependant, la relation entre l activité automatique observée sur la VP du Rat et la FA reste encore à établir.The mechanisms involved in the activation of ectopic foci within the thoracic veins, cause of atrial fibrillation (AF) in man, are still unknown. We shown that norepinephrine (NE) can induce an automatic activity in pulmonary vein (PV) but not in left atrium (LA) of the Rat. In this study, we shown: 1) a depolarised resting membrane potential in PV and superior vena cava (SVC) compared with LA and the occurence of an automatic activity in repetitive bursts in PV; 2) a depolarisation due to a1-adrenergic receptors (a1-AR) stimulation more pronounced in PV than the other two tissues, which induce an inexcitability of the PV and the SVC; 3) the occurence of a triggered activity facilitated by the simulation of b1-AR in SVC; 4) a decrease with age of the incidence of the catecholaminergic automatic activity and responses to the stimulation of a1-AR in PV and the occurence of a triggered activity under stimulation of a1-adrenergic in PV of old animals. Therefore, we have shown some functionnal differences between the myocardium of thoracic veins and atrial tissu. Nevertheless, the relationship between automatic activity observed in Rat PV and AF remains to establish.TOURS-Bibl.électronique (372610011) / SudocSudocFranceF

0151 : In vivo modulation of the cardiac Na+ current using AAVs

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In vivo Dominant-Negative Effect of an SCN5A Brugada Syndrome Variant

International audienceLoss-of-function mutations in the cardiac Na+ channel α-subunit Nav1.5, encoded by SCN5A, cause Brugada syndrome (BrS), a hereditary disease characterized by sudden cardiac death due to ventricular fibrillation. We previously evidenced in vitro the dominant-negative effect of the BrS Nav1.5-R104W variant, inducing retention of wild-type (WT) channels and leading to a drastic reduction of the resulting Na+ current (INa). To explore this dominant-negative effect in vivo, we created a murine model using adeno-associated viruses (AAVs).Methods: Due to the large size of SCN5A, a dual AAV vector strategy was used combining viral DNA recombination and trans-splicing. Mice were injected with two AAV serotypes capsid 9: one packaging the cardiac specific troponin-T promoter, the 5′ half of hSCN5A cDNA, a splicing donor site and a recombinogenic sequence; and another packaging the complementary recombinogenic sequence, a splicing acceptor site, the 3′ half of hSCN5A cDNA fused to the gfp gene sequence, and the SV40 polyA signal. Eight weeks after AAV systemic injection in wild-type (WT) mice, echocardiography and ECG were recorded and mice were sacrificed. The full-length hSCN5A-gfp expression was assessed by western blot and immunohistochemistry in transduced heart tissues and the Na+ current was recorded by the patch-clamp technique in isolated adult GFP-expressing heart cells.Results: Almost 75% of the cardiomyocytes were transduced in hearts of mice injected with hNav1.5 and ∼30% in hNav1.5-R104W overexpressing tissues. In ventricular mice cardiomyocytes expressing R104W mutant channels, the endogenous INa was significantly decreased. Moreover, overexpression of R104W channels in normal hearts led to a decrease of total Nav1.5 expression. The R104W mutant also induced a slight dilatation of mice left ventricles and a prolongation of RR interval and P-wave duration in transduced mice. Altogether, our results demonstrated an in vivo dominant-negative effect of defective R104W channels on endogenous ones.Conclusion: Using a trans-splicing and viral DNA recombination strategy to overexpress the Na+ channel in mouse hearts allowed us to demonstrate in vivo the dominant-negative effect of a BrS variant identified in the N-terminus of Nav1.5

Microtubule polymerization state and clathrin-dependent internalization regulate dynamics of cardiac potassium channel

International audienceIon channel trafficking powerfully influences cardiac electrical activity as it regulates the number of available channels at the plasma membrane. Studies have largely focused on identifying the molecular determinants of the trafficking of the atria-specific KV1.5 channel, the molecular basis of the ultra-rapid delayed rectifier current IKur. Besides, regulated KV1.5 channel recycling upon changes in homeostatic state and mechanical constraints in native cardiomyocytes has been well documented. Here, using cutting-edge imaging in live myocytes, we investigated the dynamics of this channel in the plasma membrane. We demonstrate that the clathrin pathway is a major regulator of the functional expression of KV1.5 channels in atrial myocytes, with the microtubule network as the prominent organizer of KV1.5 transport within the membrane. Both clathrin blockade and microtubule disruption result in channel clusterization with reduced membrane mobility and internalization, whereas disassembly of the actin cytoskeleton does not. Mobile KV1.5 channels are associated with the microtubule plus-end tracking protein EB1 whereas static KV1.5 clusters are associated with stable acetylated microtubules. In human biopsies from patients in atrial fibrillation associated with atrial remodeling, drastic modifications in the trafficking balance occurs together with alteration in microtubule polymerization state resulting in modest reduced endocytosis and increased recycling. Consequently, hallmark of atrial KV1.5 dynamics within the membrane is clathrin- and microtubule- dependent. During atrial remodeling, predominance of anterograde trafficking activity over retrograde trafficking could result in accumulation ok KV1.5 channels in the plasma membrane

Synaptic restoration by cAMP/PKA drives activity-dependent neuroprotection to motoneurons in ALS

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