Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia.
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.
Affiliation: Klinikum rechts der Isar, Technische Universität München, I. Medizinische Klinik, Kardiologie, München, Germany.Show MeSH
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Mentions: To direct iPSCs into the cardiac lineage, we used the EB differentiation system as previously described (Moretti et al, 2010a, b). Spontaneously beating areas, which started to appear within 10–12 days, were manually explanted and allowed to further mature for additional 2–4 months. Quantitative real-time PCR (qRT-PCR) in cardiac explants revealed that, after 2 months maturation, expression of most genes involved in myocytic Ca2+ handling and excitation–contraction (EC) coupling reaches similar levels to those of fetal human heart and is comparable among different control and CPVT-iPSC clones (Fig 2A). Consistent with already reported transcriptional profile data on human iPSC-/ESC-derived cardiac explants (Gupta et al, 2010), calsequestrin (CASQ2) expression is almost undetectable in all iPSC-derived cardiac explants at this maturation stage, while RYR2 is already expressed. However, protein analysis by western blotting at 3–4 months maturation demonstrated similar expression levels of pivotal Ca2+ handling proteins, such as RYR2, CASQ2, triadin (TRDN), junctin (JCTN) and phospholamban (PLN), among control-, CPVT-iPSC-derived cardiomyocytes and adult human heart tissue, suggesting further development of Ca2+ cycling molecular components (Fig 2B). Moreover, we evaluated the expression and subcellular localization of RYR2 by confocal immunofluorescence analysis on single control and diseased iPSC-derived cardiomyocytes (Fig 2C). In both cells, RYR2 was similarly distributed in the cytosol and perinuclear region, and partially co-localized with the myofilaments (Fig 2C). Importantly, RYR2 spatial cluster density was also comparable in control and CPVT myocytes (Fig 2D), suggesting that the S406L mutation does not interfere with trafficking of the homotetrameric channel.
Affiliation: Klinikum rechts der Isar, Technische Universität München, I. Medizinische Klinik, Kardiologie, München, Germany.