In vivo Dominant-Negative Effect of an SCN5A Brugada Syndrome Variant
Loss-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 va...
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doaj-e0b8b90bb7894a19b2b200b263c747202021-05-28T14:22:10ZengFrontiers Media S.A.Frontiers in Physiology1664-042X2021-05-011210.3389/fphys.2021.661413661413In vivo Dominant-Negative Effect of an SCN5A Brugada Syndrome VariantNicolas Doisne0Nicolas Doisne1Marta Grauso2Marta Grauso3Nathalie Mougenot4Nathalie Mougenot5Nathalie Mougenot6Michel Clergue7Michel Clergue8Charlotte Souil9Charlotte Souil10Alain Coulombe11Alain Coulombe12Pascale Guicheney13Pascale Guicheney14Nathalie Neyroud15Nathalie Neyroud16INSERM, UMR_S 1166 ICAN, Paris, FranceUMR_S 1166, Faculté de Médecine Pitié-Salpêtrière, Sorbonne Université, Paris, FranceINSERM, UMR_S 1166 ICAN, Paris, FranceUMR_S 1166, Faculté de Médecine Pitié-Salpêtrière, Sorbonne Université, Paris, FranceINSERM, UMR_S 1166 ICAN, Paris, FranceUMR_S 1166, Faculté de Médecine Pitié-Salpêtrière, Sorbonne Université, Paris, FranceUMS_28, Sorbonne Université, Paris, FranceINSERM, UMR_S 1166 ICAN, Paris, FranceUMR_S 1166, Faculté de Médecine Pitié-Salpêtrière, Sorbonne Université, Paris, FranceINSERM, UMR_S 1166 ICAN, Paris, FranceUMR_S 1166, Faculté de Médecine Pitié-Salpêtrière, Sorbonne Université, Paris, FranceINSERM, UMR_S 1166 ICAN, Paris, FranceUMR_S 1166, Faculté de Médecine Pitié-Salpêtrière, Sorbonne Université, Paris, FranceINSERM, UMR_S 1166 ICAN, Paris, FranceUMR_S 1166, Faculté de Médecine Pitié-Salpêtrière, Sorbonne Université, Paris, FranceINSERM, UMR_S 1166 ICAN, Paris, FranceUMR_S 1166, Faculté de Médecine Pitié-Salpêtrière, Sorbonne Université, Paris, FranceLoss-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).MethodsDue 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.ResultsAlmost 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.ConclusionUsing 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.https://www.frontiersin.org/articles/10.3389/fphys.2021.661413/fullBrugada syndromeNav1.5SCN5Aanimal modelelectrophysiologyAAV |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Nicolas Doisne Nicolas Doisne Marta Grauso Marta Grauso Nathalie Mougenot Nathalie Mougenot Nathalie Mougenot Michel Clergue Michel Clergue Charlotte Souil Charlotte Souil Alain Coulombe Alain Coulombe Pascale Guicheney Pascale Guicheney Nathalie Neyroud Nathalie Neyroud |
spellingShingle |
Nicolas Doisne Nicolas Doisne Marta Grauso Marta Grauso Nathalie Mougenot Nathalie Mougenot Nathalie Mougenot Michel Clergue Michel Clergue Charlotte Souil Charlotte Souil Alain Coulombe Alain Coulombe Pascale Guicheney Pascale Guicheney Nathalie Neyroud Nathalie Neyroud In vivo Dominant-Negative Effect of an SCN5A Brugada Syndrome Variant Frontiers in Physiology Brugada syndrome Nav1.5 SCN5A animal model electrophysiology AAV |
author_facet |
Nicolas Doisne Nicolas Doisne Marta Grauso Marta Grauso Nathalie Mougenot Nathalie Mougenot Nathalie Mougenot Michel Clergue Michel Clergue Charlotte Souil Charlotte Souil Alain Coulombe Alain Coulombe Pascale Guicheney Pascale Guicheney Nathalie Neyroud Nathalie Neyroud |
author_sort |
Nicolas Doisne |
title |
In vivo Dominant-Negative Effect of an SCN5A Brugada Syndrome Variant |
title_short |
In vivo Dominant-Negative Effect of an SCN5A Brugada Syndrome Variant |
title_full |
In vivo Dominant-Negative Effect of an SCN5A Brugada Syndrome Variant |
title_fullStr |
In vivo Dominant-Negative Effect of an SCN5A Brugada Syndrome Variant |
title_full_unstemmed |
In vivo Dominant-Negative Effect of an SCN5A Brugada Syndrome Variant |
title_sort |
in vivo dominant-negative effect of an scn5a brugada syndrome variant |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Physiology |
issn |
1664-042X |
publishDate |
2021-05-01 |
description |
Loss-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).MethodsDue 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.ResultsAlmost 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.ConclusionUsing 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. |
topic |
Brugada syndrome Nav1.5 SCN5A animal model electrophysiology AAV |
url |
https://www.frontiersin.org/articles/10.3389/fphys.2021.661413/full |
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