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Calcium signaling consequences of RyR2 mutations associated with CPVT1 introduced via CRISPR/Cas9 gene editing in human-induced pluripotent stem cell–derived cardiomyocytes: Comparison of RyR2-R420Q, F2483I, and Q4201R

  • Xiao-Hua Zhang
    Affiliations
    Cardiac Signaling Center of University of South Carolina, Medical University of South Carolina, and Clemson University, Charleston, South Carolina
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  • Hua Wei
    Affiliations
    Cardiac Signaling Center of University of South Carolina, Medical University of South Carolina, and Clemson University, Charleston, South Carolina
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  • Yanli Xia
    Affiliations
    Cardiac Signaling Center of University of South Carolina, Medical University of South Carolina, and Clemson University, Charleston, South Carolina
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  • Martin Morad
    Correspondence
    Address reprint requests and correspondence: Dr. Martin Morad, Cardiac Signaling Center of University of South Carolina, Medical University of South Carolina, and Clemson University, 68 President St, Bioengineering Building, Room 306, Charleston, SC 29403.
    Affiliations
    Cardiac Signaling Center of University of South Carolina, Medical University of South Carolina, and Clemson University, Charleston, South Carolina
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Published:September 12, 2020DOI:https://doi.org/10.1016/j.hrthm.2020.09.007

      Background

      Human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) created from patients with catecholaminergic polymorphic ventricular tachycardia 1 (CPVT1) have been used to study CPVT1 arrhythmia.

      Objective

      The purpose of this study was to evaluate the Ca2+ signaling aberrancies and pharmacological sensitivities of 3 CRISPR/Cas9-introduced CPVT1 mutations located in different molecular domains of ryanodine receptor 2 (RyR2).

      Methods

      CRISPR/Cas9-engineered hiPSC-CMs carrying RyR2 mutations—R420Q, Q4201R, and F2483I—were voltage clamped, and their electrophysiology, pharmacology, and Ca2+ signaling phenotypes measured using total internal reflection fluorescence microscopy.

      Results

      R420Q and Q4201R mutant hiPSC-CMs exhibit irregular, long-lasting, spatially wandering Ca2+ sparks and aberrant Ca2+ releases similar to F2483I unlike the wild-type myocytes. Large sarcoplasmic reticulum (SR) Ca2+ leaks and smaller SR Ca2+ contents were detected in cells expressing Q4201R and F2483I, but not R420Q. Fractional Ca2+ release and calcium-induced calcium release gain were higher in Q4201R than in R420Q and F2483I hiPSC-CMs. JTV519 was equally effective in suppressing Ca2+ sparks, waves, and SR Ca2+ leaks in hiPSC-CMs derived from all 3 mutant lines. Flecainide and dantrolene similarly suppressed SR Ca2+ leaks, but were less effective in decreasing spark frequency and durations.

      Conclusion

      CRISPR/Cas9 gene editing of hiPSCs provides a novel approach in studying CPVT1-associated RyR2 mutations and suggests that Ca2+-signaling aberrancies and drug sensitivities may vary depending on the mutation site.

      Graphical abstract

      Keywords

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