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Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia.

Jung CB, Moretti A, Mederos y Schnitzler M, Iop L, Storch U, Bellin M, Dorn T, Ruppenthal S, Pfeiffer S, Goedel A, Dirschinger RJ, Seyfarth M, Lam JT, Sinnecker D, Gudermann T, Lipp P, Laugwitz KL - EMBO Mol Med (2012)

Bottom Line: In patient iPSC-derived cardiomyocytes, catecholaminergic stress led to elevated diastolic Ca(2+) concentrations, a reduced SR Ca(2+) content and an increased susceptibility to DADs and arrhythmia as compared to control myocytes.Dantrolene, a drug effective on malignant hyperthermia, restored normal Ca(2+) spark properties and rescued the arrhythmogenic phenotype.This suggests defective inter-domain interactions within the RYR2 channel as the pathomechanism of the S406L mutation.

View Article: PubMed Central - PubMed

Affiliation: Klinikum rechts der Isar, Technische Universität München, I. Medizinische Klinik, Kardiologie, München, Germany.

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Generation of CPVT-iPSCsPedigree of the CPVT-affected patient (III-1) showing autosomal dominant inheritance in the family.Sequence analysis of RYR2 gene in fibroblasts from control and CPVT patient, revealing a novel heterozygous missense mutation in exon 14 (position 1217C > T of the coding sequence). Same results were obtained from all analysed control and CPVT-iPSC clones.Schematic representation of RYR2 channel and localization of the S406L mutation (red circle) at the N-terminal domain. Yellow circles indicate reported putative pathogenic mutations.Representative images of CPVT-iPSC colonies in bright field (top, clone a) and after staining for alkaline phosphatase (AP) activity (bottom, clone c). Scale bars, 100 µm.Representative images of a CPVT-iPSC colony (clone b) after immunostaining for the pluripotency markers NANOG (red) and TRA1-81 (green). Merged image is the magnified area marked by the white box. Scale bars, 100 µm.Karyogram of CPVT-iPSC clone a.
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fig01: Generation of CPVT-iPSCsPedigree of the CPVT-affected patient (III-1) showing autosomal dominant inheritance in the family.Sequence analysis of RYR2 gene in fibroblasts from control and CPVT patient, revealing a novel heterozygous missense mutation in exon 14 (position 1217C > T of the coding sequence). Same results were obtained from all analysed control and CPVT-iPSC clones.Schematic representation of RYR2 channel and localization of the S406L mutation (red circle) at the N-terminal domain. Yellow circles indicate reported putative pathogenic mutations.Representative images of CPVT-iPSC colonies in bright field (top, clone a) and after staining for alkaline phosphatase (AP) activity (bottom, clone c). Scale bars, 100 µm.Representative images of a CPVT-iPSC colony (clone b) after immunostaining for the pluripotency markers NANOG (red) and TRA1-81 (green). Merged image is the magnified area marked by the white box. Scale bars, 100 µm.Karyogram of CPVT-iPSC clone a.

Mentions: Dermal fibroblasts were obtained from a 24-year-old woman with a diagnosis of familial CPVT, who underwent cardiac arrest at the age of 23 years and received an ICD after cardiac resuscitation. Genetic screening showed that she carried a novel autosomal dominant S406L missense mutation in the RYR2 gene, caused by a C → T nucleotide substitution in exon 14 at position 1217 of the coding region (Fig 1A and B). The mutation is located in the N-terminal domain (amino acids 1–600) of the RYR2 Ca2+ release channel, which represents, together with the central domain (amino acids 2000–2500) and the carboxy-terminal transmembrane domain, one of the three hotspots for CPVT-associated RYR2 mutations (George et al, 2007; Thomas et al, 2010; Fig 1C). Fibroblast transduction with retroviral vectors encoding for SOX2, OCT4, KLF4 and c-MYC generated several CPVT patient-specific iPSC clones, three of which were further characterized and used for cardiomyocyte differentiation. Similarly, control iPSCs were created using fibroblasts from a 32-year-old healthy female (Moretti et al, 2010b). The S406L heterozygous mutation was identified exclusively in CPVT-iPSCs. All iPSC lines showed human embryonic stem cell morphology, expression of the pluripotency markers NANOG and TRA1-81, alkaline phosphatase activity, reactivation of endogenous pluripotency genes (OCT4, SOX2, NANOG, REX1 and TDGF1), silencing of the four retroviral transgenes and normal karyotype (Fig 1D–F; Fig S1A and B of Supporting information). Pluripotency of each iPSC line was assessed by upregulation of genes specific of all three germ layers in in vitro-differentiating embryoid bodies (EBs) (Fig S1C of Supporting information).


Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia.

Jung CB, Moretti A, Mederos y Schnitzler M, Iop L, Storch U, Bellin M, Dorn T, Ruppenthal S, Pfeiffer S, Goedel A, Dirschinger RJ, Seyfarth M, Lam JT, Sinnecker D, Gudermann T, Lipp P, Laugwitz KL - EMBO Mol Med (2012)

Generation of CPVT-iPSCsPedigree of the CPVT-affected patient (III-1) showing autosomal dominant inheritance in the family.Sequence analysis of RYR2 gene in fibroblasts from control and CPVT patient, revealing a novel heterozygous missense mutation in exon 14 (position 1217C > T of the coding sequence). Same results were obtained from all analysed control and CPVT-iPSC clones.Schematic representation of RYR2 channel and localization of the S406L mutation (red circle) at the N-terminal domain. Yellow circles indicate reported putative pathogenic mutations.Representative images of CPVT-iPSC colonies in bright field (top, clone a) and after staining for alkaline phosphatase (AP) activity (bottom, clone c). Scale bars, 100 µm.Representative images of a CPVT-iPSC colony (clone b) after immunostaining for the pluripotency markers NANOG (red) and TRA1-81 (green). Merged image is the magnified area marked by the white box. Scale bars, 100 µm.Karyogram of CPVT-iPSC clone a.
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Related In: Results  -  Collection

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fig01: Generation of CPVT-iPSCsPedigree of the CPVT-affected patient (III-1) showing autosomal dominant inheritance in the family.Sequence analysis of RYR2 gene in fibroblasts from control and CPVT patient, revealing a novel heterozygous missense mutation in exon 14 (position 1217C > T of the coding sequence). Same results were obtained from all analysed control and CPVT-iPSC clones.Schematic representation of RYR2 channel and localization of the S406L mutation (red circle) at the N-terminal domain. Yellow circles indicate reported putative pathogenic mutations.Representative images of CPVT-iPSC colonies in bright field (top, clone a) and after staining for alkaline phosphatase (AP) activity (bottom, clone c). Scale bars, 100 µm.Representative images of a CPVT-iPSC colony (clone b) after immunostaining for the pluripotency markers NANOG (red) and TRA1-81 (green). Merged image is the magnified area marked by the white box. Scale bars, 100 µm.Karyogram of CPVT-iPSC clone a.
Mentions: Dermal fibroblasts were obtained from a 24-year-old woman with a diagnosis of familial CPVT, who underwent cardiac arrest at the age of 23 years and received an ICD after cardiac resuscitation. Genetic screening showed that she carried a novel autosomal dominant S406L missense mutation in the RYR2 gene, caused by a C → T nucleotide substitution in exon 14 at position 1217 of the coding region (Fig 1A and B). The mutation is located in the N-terminal domain (amino acids 1–600) of the RYR2 Ca2+ release channel, which represents, together with the central domain (amino acids 2000–2500) and the carboxy-terminal transmembrane domain, one of the three hotspots for CPVT-associated RYR2 mutations (George et al, 2007; Thomas et al, 2010; Fig 1C). Fibroblast transduction with retroviral vectors encoding for SOX2, OCT4, KLF4 and c-MYC generated several CPVT patient-specific iPSC clones, three of which were further characterized and used for cardiomyocyte differentiation. Similarly, control iPSCs were created using fibroblasts from a 32-year-old healthy female (Moretti et al, 2010b). The S406L heterozygous mutation was identified exclusively in CPVT-iPSCs. All iPSC lines showed human embryonic stem cell morphology, expression of the pluripotency markers NANOG and TRA1-81, alkaline phosphatase activity, reactivation of endogenous pluripotency genes (OCT4, SOX2, NANOG, REX1 and TDGF1), silencing of the four retroviral transgenes and normal karyotype (Fig 1D–F; Fig S1A and B of Supporting information). Pluripotency of each iPSC line was assessed by upregulation of genes specific of all three germ layers in in vitro-differentiating embryoid bodies (EBs) (Fig S1C of Supporting information).

Bottom Line: In patient iPSC-derived cardiomyocytes, catecholaminergic stress led to elevated diastolic Ca(2+) concentrations, a reduced SR Ca(2+) content and an increased susceptibility to DADs and arrhythmia as compared to control myocytes.Dantrolene, a drug effective on malignant hyperthermia, restored normal Ca(2+) spark properties and rescued the arrhythmogenic phenotype.This suggests defective inter-domain interactions within the RYR2 channel as the pathomechanism of the S406L mutation.

View Article: PubMed Central - PubMed

Affiliation: Klinikum rechts der Isar, Technische Universität München, I. Medizinische Klinik, Kardiologie, München, Germany.

Show MeSH
Related in: MedlinePlus