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Temporal requirement of Nkx2-5 during cardiac conduction system development
Archives of Cardiovascular Disease Supplements (2018) 10, 194—195
Available online at
ScienceDirect www.sciencedirect.com
Topic 7 — Electrophysiology, rhythmology and stimulation — C April 5th, Thursday 2018 195
A loss-of-function CACNA1C variant, p.T1787M, associated with risk of ventricular fibrillation in Africans M. Blancard 1,∗ , A. Debbiche 1 , K. Kato 1 , C. Cardin 2 , S. Guichard 3 , E. Gandjbakhch 1,4 , M. Haissaguerre 5,6 , F. Extramania 7 , M. Hocini 5,6 , O. Geoffroy 8 , A. Leenhardt 7 , P. Guicheney 1 , J.S. Rougier 3 1 INSERM U1166, Paris, France 2 University hospital Rangueil, Toulouse, France 3 Institute of biochemistry and molecular medicine, university of Bern, Berne, Switzerland 4 Département de cardiologie, hôpital Pitié-Salpêtrière, AP—HP, Paris, France 5 Université de Bordeaux, l’institut de rythmologie et modélisation cardiaque (LIRYC), Bordeaux, France 6 Université de Bordeaux, Inserm U1045 CRCTB, Bordeaux, France 7 Département de cardiologie, centre de référence des maladies cardiaques héréditaires, hôpital Bichat, AP—HP, Paris, France 8 CHU Sud Réunion, Saint-Pierre, La Reunion ∗ Corresponding author. E-mail address: [email protected] (M. Blancard) Introduction Calcium regulation plays a central role in cardiac function. Several variants in the L-type calcium channel, Cav1.2, have been implicated in inherited arrhythmic disorders leading to sudden death. There is a clear need to explore the function of new variants of Cav1.2 channel to gain a better understanding of lifethreatening conditions triggered by these variants. Objective We screened a cohort of patients suffering from Brugada syndrome, short QT syndrome, early repolarization syndrome and idiopathic ventricular fibrillation to identify variants in Cav1.2 coding genes in order to determine their frequency and their functional consequences. Methods Cav1.2 related genes (CACNA1C, CACNB2 and CACNA2D1) were screened in 65 probands by high resolution melting and Sanger sequencing, or by exome sequencing to identify variants. The conservation, location and frequency of these variants were determined from genetic databases. Missense variants were introduced in Cav1.2 alpha subunit plasmids by
1878-6480/
directed mutagenesis to perform electrophysiological studies after transfection of TsA-201 cells in order to assess their pathogenicity. Results Six missense variants were identified in five individuals. Five of them, p.A1648T, p.A1689T, p.G1795R, p.R1973Q, p.C1992F, did not alter the channel function. The sixth variant, p.T1787M, was identified in two patients with resuscitated cardiac arrest. The first patient originated from Cameroon and presented with an early repolarization syndrome and the second was an inhabitant of La Reunion Island with idiopathic ventricular fibrillation originating from Purkinje tissues. Patch-clamp analysis revealed that this variant significantly reduces the barium current compared to the wild-type channel without affecting the biophysical properties of the Cav1.2 channel. Conclusion We identified a loss-of-function variant, Cav1.2T1787M, a probable risk factor for ventricular fibrillation, only present in the African population. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2018.02.042 356
Temporal requirement of Nkx2-5 during cardiac conduction system development C. Choquet ∗ , R. Kelly , L. Miquerol IBDM, Marseille, France ∗ Corresponding author. E-mail address: [email protected] (C. Choquet) During cardiac development, transient invaginations of the myocardium, termed trabeculae, appear at the inner surface of the ventricles, which disappeared by compaction to form a functional ventricular wall. Trabeculae also contain progenitor cells of the ventricular conduction system (VCS), a complex network of Purkinje fibers driving the rapid propagation of electrical activity in the ventricles. Defects in ventricular compaction and conduction have been observed in patients and mutant mice carrying mutations in NKX2-5, encoding a key transcriptional regulator of heart development. In order to analyze the link between trabecular fate and VCS differentiation, we carried out genetic tracing and prospective clonal analyses of trabeculae using Cx40-CreERT2 mice and Rosa26-Confetti mice. Our results show that the peripheral VCS segregates progressively during embryonic development. Cells exclusively fated to give rise to the VCS are present in the trabe-
Topic 7 — Electrophysiology, rhythmology and stimulation — C cular compartment as early as E9.5. Specification of new conductive myocytes within the trabecular compartment during subsequent development contributes to the formation of a complex Purkinje fiber network at birth. We performed the same lineage tracing experiments in Nkx2-5 heterozygous mice with severe hypoplasia of the Purkinje fiber network at adult stages. The number of Purkinje fibers originating from early Nkx2-5 hapolinsufficient progenitors is identical to control. In contrast, at later stages of development, we observed an increase in the number of trabecular cells that do not give rise to Purkinje fibers. This suggests that Nkx2-5 controls the progressive recruitment of trabecular cells into the VCS as well as the later maintenance of the conductive phenotype. Overall our study highlights the early segregation of the VCS lineage at the onset of trabeculation and the later role of Nkx2-5 in formation of the Purkinje fiber network. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2018.02.043 249
RRAD mutation causes electrical and cytoskeletal defects in cardiomyocytes derived from a familial case of Brugada syndrome N. Belbachir 1,∗ , V. Portero 1 , J.B. Gourraud 1 , L. Jesel 2 , C. Guilluy 3 , N. Gaborit 4 , A. Girardeau 4 , S. Bonnaud 4 , S. Pattier 1 , C. Scott 5 , S. Burel 4 , A. Gaignerie 6 , E. Genin 7 , J.F. Deleuze 8 , C. Dina 4 , J.J. Schott 4 , V. Probst 1 , R. Redon 4 , F. Charpentier 4 , S. Le Scouarnec 4 1 L’institut du thorax INSERM, CNRS, université Nantes, Nantes, France 2 Service de cardiologie, CHU de Strasbourg, Strasbourg, France 3 Institute for Advanced Biosciences, INSERM, CNRS, Grenoble, France 4 Service de cardiologie, l’institut du thorax, CHU de Nantes, Nantes, France 5 The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK 6 INSERM, CNRS, UNIV Nantes, CHU Nantes, SFR Franc ¸ois Bonamy, iPSC core facility, Nantes, France 7 Inserm UMR-1078, university Brest, CHRU Brest, Brest, France 8 Centre national de recherche en génomique humaine, institut de génomique, CEA, Evry, France ∗ Corresponding author. E-mail address: [email protected] (N. Belbachir) Introduction The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias and sudden death. In the present day, only 30% of BrS cases have known genetic causes. Using whole-exome sequencing in a large pedigree with affected members, we identified a rare variant (p.R211H) in RRAD, the gene encoding Rad GTPase. Objective The aim of this work was to elucidate the mechanisms by which the RRAD p.R211H variant leads to BrS. Methods The study was performed in two cell models, i.e., cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) reprogrammed from the proband carrying the familial mutation and his healthy brother (non-carrier) and neonatal mouse cardiomyocytes (NMC) infected with adenoviruses encoding wildtype human RAD (ad WT-RAD) or GFP (ad GFP), and knock-in mouse mo del carrying the equivalent p.R210H Rad mutation (KI mice). Results iPSC-CMs from the proband displayed a severe decrease of INa and a moderate decrease of ICaL. Overexpression of WT-RAD in NMC triggered similar impact on both currents. The iPSC-CMs carrying the variant exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations. Combined with the electrical phenotype, these cells showed cortical distribution of actin, cell
195 rounding and reduced focal adhesion count. In mice, Rad was predominantly expressed in the right ventricle outflow tract compared to the other cardiac compartments. This pattern of expression was not altered by the variant. KI mice displayed ventricular conduction disorders under ajmaline challenge and histological anomalies, including fibrosis development, in the right ventricular wall. Conclusion Mutation in the RAD GTPase recapitulate the typical electrophysiological signature of Brugada syndrome coupled with cytoskeleton disturbances in cardiomyocytes derived from the patient. KI mice showed both cardiac electrical and structural disturbances. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2018.02.044 205
Low and high cardiac FKBP12.6 overexpression are associated with distinct phenotypes M. Gandon-Renard 1,∗ , F. Lefebvre 1 , P. Gerbaud 1 , P. Rouet-Benzineb 2 , A.M. Gomez 1 , J.J. Mercadier 1 1 INSERM UMR-S 1180, Châtenay-Malabry, France 2 INSERM UMR-S 942, Paris, France ∗ Corresponding author. E-mail address: [email protected] (M. Gandon-Renard) The ryanodine receptors RyR2 have a key role in cardiac excitationcontraction coupling (ECC) and are regulated, among others, by FK506 binding proteins (FKBP). FKBP12 is more abundant than FKBP12.6 in cardiomyocytes with a low affinity for RyR2, whereas FKBP12.6 with stronger affinity is less abundant. In heart failure, FKBP expression decreases and RyR2 are hyperphosphorylated, both mechanisms involved in arrhythmia development. Cardiac FKBP12.6 overexpression protects against arrhythmias due to ß-adrenergic stimulation, but this mechanism is still unknown. Our hypothesis is that FKBP12.6 overexpression, with its stronger affinity for RyR2, displaces FKBP12, therefore reducing RyR2 sensitivity to sympathetic stimulation. To address this issue, we developed 2 transgenic mouse lines with low (TG1) and high (TG2) levels of cardiac-specific FKBP12.6 overexpression. We characterized ECC of both lines in basal conditions and after ß-adrenergic stimulation to confirm the anti-adrenergic effects of FKBP12.6 overexpression in link with the expression levels and the stoichiometry of the 2 FKBP isoforms. ECC was characterized by confocal microscopy and expression levels of both isoforms by Western blot with commercial and homemade antibodies. Our first results showed that TG1 mice had normal cardiac phenotype, whereas TG2 mice exhibited cardiac hypertrophy. In TG1 mice, basal Ca2+ transient (Catr) amplitude was slightly higher than that in wild type mice (WT), while time constant of Catr decay was unchanged, the latter being slower in TG2 mice. When 50 nM isoproterenol was added, the increase of Catr amplitude was smaller in TG1 (31%) than in WT (50%) and TG2 (60%) mice. Similarly, the acceleration of Catr decay was smaller in TG1 than in TG2 mice (−27% vs. −56%, respectively). These preliminary results suggest that low FKBP12.6 overexpression protects partially against sympathetic stimulation, whereas high FKBP12.6 overexpression is associated with cardiomyopathy development. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2018.02.045
Available online at
ScienceDirect www.sciencedirect.com
Topic 7 — Electrophysiology, rhythmology and stimulation — C April 5th, Thursday 2018 195
A loss-of-function CACNA1C variant, p.T1787M, associated with risk of ventricular fibrillation in Africans M. Blancard 1,∗ , A. Debbiche 1 , K. Kato 1 , C. Cardin 2 , S. Guichard 3 , E. Gandjbakhch 1,4 , M. Haissaguerre 5,6 , F. Extramania 7 , M. Hocini 5,6 , O. Geoffroy 8 , A. Leenhardt 7 , P. Guicheney 1 , J.S. Rougier 3 1 INSERM U1166, Paris, France 2 University hospital Rangueil, Toulouse, France 3 Institute of biochemistry and molecular medicine, university of Bern, Berne, Switzerland 4 Département de cardiologie, hôpital Pitié-Salpêtrière, AP—HP, Paris, France 5 Université de Bordeaux, l’institut de rythmologie et modélisation cardiaque (LIRYC), Bordeaux, France 6 Université de Bordeaux, Inserm U1045 CRCTB, Bordeaux, France 7 Département de cardiologie, centre de référence des maladies cardiaques héréditaires, hôpital Bichat, AP—HP, Paris, France 8 CHU Sud Réunion, Saint-Pierre, La Reunion ∗ Corresponding author. E-mail address: [email protected] (M. Blancard) Introduction Calcium regulation plays a central role in cardiac function. Several variants in the L-type calcium channel, Cav1.2, have been implicated in inherited arrhythmic disorders leading to sudden death. There is a clear need to explore the function of new variants of Cav1.2 channel to gain a better understanding of lifethreatening conditions triggered by these variants. Objective We screened a cohort of patients suffering from Brugada syndrome, short QT syndrome, early repolarization syndrome and idiopathic ventricular fibrillation to identify variants in Cav1.2 coding genes in order to determine their frequency and their functional consequences. Methods Cav1.2 related genes (CACNA1C, CACNB2 and CACNA2D1) were screened in 65 probands by high resolution melting and Sanger sequencing, or by exome sequencing to identify variants. The conservation, location and frequency of these variants were determined from genetic databases. Missense variants were introduced in Cav1.2 alpha subunit plasmids by
1878-6480/
directed mutagenesis to perform electrophysiological studies after transfection of TsA-201 cells in order to assess their pathogenicity. Results Six missense variants were identified in five individuals. Five of them, p.A1648T, p.A1689T, p.G1795R, p.R1973Q, p.C1992F, did not alter the channel function. The sixth variant, p.T1787M, was identified in two patients with resuscitated cardiac arrest. The first patient originated from Cameroon and presented with an early repolarization syndrome and the second was an inhabitant of La Reunion Island with idiopathic ventricular fibrillation originating from Purkinje tissues. Patch-clamp analysis revealed that this variant significantly reduces the barium current compared to the wild-type channel without affecting the biophysical properties of the Cav1.2 channel. Conclusion We identified a loss-of-function variant, Cav1.2T1787M, a probable risk factor for ventricular fibrillation, only present in the African population. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2018.02.042 356
Temporal requirement of Nkx2-5 during cardiac conduction system development C. Choquet ∗ , R. Kelly , L. Miquerol IBDM, Marseille, France ∗ Corresponding author. E-mail address: [email protected] (C. Choquet) During cardiac development, transient invaginations of the myocardium, termed trabeculae, appear at the inner surface of the ventricles, which disappeared by compaction to form a functional ventricular wall. Trabeculae also contain progenitor cells of the ventricular conduction system (VCS), a complex network of Purkinje fibers driving the rapid propagation of electrical activity in the ventricles. Defects in ventricular compaction and conduction have been observed in patients and mutant mice carrying mutations in NKX2-5, encoding a key transcriptional regulator of heart development. In order to analyze the link between trabecular fate and VCS differentiation, we carried out genetic tracing and prospective clonal analyses of trabeculae using Cx40-CreERT2 mice and Rosa26-Confetti mice. Our results show that the peripheral VCS segregates progressively during embryonic development. Cells exclusively fated to give rise to the VCS are present in the trabe-
Topic 7 — Electrophysiology, rhythmology and stimulation — C cular compartment as early as E9.5. Specification of new conductive myocytes within the trabecular compartment during subsequent development contributes to the formation of a complex Purkinje fiber network at birth. We performed the same lineage tracing experiments in Nkx2-5 heterozygous mice with severe hypoplasia of the Purkinje fiber network at adult stages. The number of Purkinje fibers originating from early Nkx2-5 hapolinsufficient progenitors is identical to control. In contrast, at later stages of development, we observed an increase in the number of trabecular cells that do not give rise to Purkinje fibers. This suggests that Nkx2-5 controls the progressive recruitment of trabecular cells into the VCS as well as the later maintenance of the conductive phenotype. Overall our study highlights the early segregation of the VCS lineage at the onset of trabeculation and the later role of Nkx2-5 in formation of the Purkinje fiber network. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2018.02.043 249
RRAD mutation causes electrical and cytoskeletal defects in cardiomyocytes derived from a familial case of Brugada syndrome N. Belbachir 1,∗ , V. Portero 1 , J.B. Gourraud 1 , L. Jesel 2 , C. Guilluy 3 , N. Gaborit 4 , A. Girardeau 4 , S. Bonnaud 4 , S. Pattier 1 , C. Scott 5 , S. Burel 4 , A. Gaignerie 6 , E. Genin 7 , J.F. Deleuze 8 , C. Dina 4 , J.J. Schott 4 , V. Probst 1 , R. Redon 4 , F. Charpentier 4 , S. Le Scouarnec 4 1 L’institut du thorax INSERM, CNRS, université Nantes, Nantes, France 2 Service de cardiologie, CHU de Strasbourg, Strasbourg, France 3 Institute for Advanced Biosciences, INSERM, CNRS, Grenoble, France 4 Service de cardiologie, l’institut du thorax, CHU de Nantes, Nantes, France 5 The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK 6 INSERM, CNRS, UNIV Nantes, CHU Nantes, SFR Franc ¸ois Bonamy, iPSC core facility, Nantes, France 7 Inserm UMR-1078, university Brest, CHRU Brest, Brest, France 8 Centre national de recherche en génomique humaine, institut de génomique, CEA, Evry, France ∗ Corresponding author. E-mail address: [email protected] (N. Belbachir) Introduction The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias and sudden death. In the present day, only 30% of BrS cases have known genetic causes. Using whole-exome sequencing in a large pedigree with affected members, we identified a rare variant (p.R211H) in RRAD, the gene encoding Rad GTPase. Objective The aim of this work was to elucidate the mechanisms by which the RRAD p.R211H variant leads to BrS. Methods The study was performed in two cell models, i.e., cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) reprogrammed from the proband carrying the familial mutation and his healthy brother (non-carrier) and neonatal mouse cardiomyocytes (NMC) infected with adenoviruses encoding wildtype human RAD (ad WT-RAD) or GFP (ad GFP), and knock-in mouse mo del carrying the equivalent p.R210H Rad mutation (KI mice). Results iPSC-CMs from the proband displayed a severe decrease of INa and a moderate decrease of ICaL. Overexpression of WT-RAD in NMC triggered similar impact on both currents. The iPSC-CMs carrying the variant exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations. Combined with the electrical phenotype, these cells showed cortical distribution of actin, cell
195 rounding and reduced focal adhesion count. In mice, Rad was predominantly expressed in the right ventricle outflow tract compared to the other cardiac compartments. This pattern of expression was not altered by the variant. KI mice displayed ventricular conduction disorders under ajmaline challenge and histological anomalies, including fibrosis development, in the right ventricular wall. Conclusion Mutation in the RAD GTPase recapitulate the typical electrophysiological signature of Brugada syndrome coupled with cytoskeleton disturbances in cardiomyocytes derived from the patient. KI mice showed both cardiac electrical and structural disturbances. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2018.02.044 205
Low and high cardiac FKBP12.6 overexpression are associated with distinct phenotypes M. Gandon-Renard 1,∗ , F. Lefebvre 1 , P. Gerbaud 1 , P. Rouet-Benzineb 2 , A.M. Gomez 1 , J.J. Mercadier 1 1 INSERM UMR-S 1180, Châtenay-Malabry, France 2 INSERM UMR-S 942, Paris, France ∗ Corresponding author. E-mail address: [email protected] (M. Gandon-Renard) The ryanodine receptors RyR2 have a key role in cardiac excitationcontraction coupling (ECC) and are regulated, among others, by FK506 binding proteins (FKBP). FKBP12 is more abundant than FKBP12.6 in cardiomyocytes with a low affinity for RyR2, whereas FKBP12.6 with stronger affinity is less abundant. In heart failure, FKBP expression decreases and RyR2 are hyperphosphorylated, both mechanisms involved in arrhythmia development. Cardiac FKBP12.6 overexpression protects against arrhythmias due to ß-adrenergic stimulation, but this mechanism is still unknown. Our hypothesis is that FKBP12.6 overexpression, with its stronger affinity for RyR2, displaces FKBP12, therefore reducing RyR2 sensitivity to sympathetic stimulation. To address this issue, we developed 2 transgenic mouse lines with low (TG1) and high (TG2) levels of cardiac-specific FKBP12.6 overexpression. We characterized ECC of both lines in basal conditions and after ß-adrenergic stimulation to confirm the anti-adrenergic effects of FKBP12.6 overexpression in link with the expression levels and the stoichiometry of the 2 FKBP isoforms. ECC was characterized by confocal microscopy and expression levels of both isoforms by Western blot with commercial and homemade antibodies. Our first results showed that TG1 mice had normal cardiac phenotype, whereas TG2 mice exhibited cardiac hypertrophy. In TG1 mice, basal Ca2+ transient (Catr) amplitude was slightly higher than that in wild type mice (WT), while time constant of Catr decay was unchanged, the latter being slower in TG2 mice. When 50 nM isoproterenol was added, the increase of Catr amplitude was smaller in TG1 (31%) than in WT (50%) and TG2 (60%) mice. Similarly, the acceleration of Catr decay was smaller in TG1 than in TG2 mice (−27% vs. −56%, respectively). These preliminary results suggest that low FKBP12.6 overexpression protects partially against sympathetic stimulation, whereas high FKBP12.6 overexpression is associated with cardiomyopathy development. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2018.02.045