Characterization of N-terminal RYR2 variants outside CPVT1 hotspot regions using patient iPSCs reveal pathogenesis and therapeutic potential

Marissa J. Stutzman, C. S.John Kim, David J. Tester, Samantha K. Hamrick, Steven M. Dotzler, John R. Giudicessi, Marco C. Miotto, Jeevan B. Gc, Joachim Frank, Andrew R. Marks, Michael John Ackerman

Research output: Contribution to journalArticlepeer-review

Abstract

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a cardiac channelopathy causing ventricular tachycardia following adrenergic stimulation. Pathogenic variants in RYR2-encoded ryanodine receptor 2 (RYR2) cause CPVT1 and cluster into domains I-IV, with the most N-terminal domain involving residues 77-466. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated for RYR2-F13L, -L14P, -R15P, and -R176Q variants. Isogenic control iPSCs were generated using CRISPR-Cas9/PiggyBac. Fluo-4 Ca2+ imaging assessed Ca2+ handling with/without isoproterenol (ISO), nadolol (Nad), and flecainide (Flec) treatment. CPVT1 iPSC-CMs displayed increased Ca2+ sparking and Ca2+ transient amplitude following ISO compared with control. Combined Nad treatment/ISO stimulation reduced Ca2+ amplitude and sparking in variant iPSC-CMs. Molecular dynamic simulations visualized the structural role of these variants. We provide the first functional evidence that these most proximal N-terminal localizing variants alter calcium handling similar to CPVT1. These variants are located at the N-terminal domain and the central domain interface and could destabilize the RYR2 channel promoting Ca2+ leak-triggered arrhythmias.

Original languageEnglish (US)
Pages (from-to)2023-2036
Number of pages14
JournalStem Cell Reports
Volume17
Issue number9
DOIs
StatePublished - Sep 13 2022

Keywords

  • calcium handling
  • catecholaminergic polymorphic ventricular tachycardia
  • induced pluripotent stem cell-derived cardiomyocytes
  • ryanodine receptor
  • sudden cardiac death

ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Developmental Biology
  • Cell Biology

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