Abstract
Human-induced pluripotent stem cells (hiPSCs) provide a powerful platform to study biophysical and molecular mechanisms underlying the pathophysiology of genetic mutations associated with cardiac arrhythmia. Human iPSCs can be generated by reprograming of dermal fibroblasts of normal or diseased individuals and be differentiated into cardiac myocytes. Obtaining biopsies from patients afflicted with point mutations causing arrhythmia is often a cumbersome process even when patients are available. Recent development of CRISPR/Cas9 gene editing system makes it, however, possible to introduce arrhythmia-associated point mutations at the desired loci of the wild-type hiPSCs in relatively short times. This platform was used by us to compare the Ca2+ signaling phenotypes of cardiomyocytes harboring point mutations in cardiac Ca2+ release channel, type-2 ryanodine receptor (RyR2), since over 200 missense mutations in RYR2 gene appear to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1). We have created cardiac myocytes harboring mutations in different domains of RyR2, to study not only their Ca2+ signaling consequences but also their drug and domain specificity as related to CPVT1 pathology. In this chapter, we describe our procedures to establish CRISPR/Cas9 gene-edited hiPSC-derived cardiomyocytes.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Napolitano C, Bloise R, Monteforte N, Priori SG (2012) Sudden cardiac death and genetic ion channelopathies: long QT, Brugada, short QT, catecholaminergic polymorphic ventricular tachycardia, and idiopathic ventricular fibrillation. Circulation 125(16):2027–2034. https://doi.org/10.1161/CIRCULATIONAHA.111.055947
Kushnir A, Wajsberg B, Marks AR (2018) Ryanodine receptor dysfunction in human disorders. Biochim Biophys Acta Mol Cell Res 1865(11 Pt B):1687–1697. https://doi.org/10.1016/j.bbamcr.2018.07.011
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872. https://doi.org/10.1016/j.cell.2007.11.019
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308. https://doi.org/10.1038/nprot.2013.143
Wei H, Zhang XH, Clift C, Yamaguchi N, Morad M (2018) CRISPR/Cas9 Gene editing of RyR2 in human stem cell-derived cardiomyocytes provides a novel approach in investigating dysfunctional Ca(2+) signaling. Cell Calcium 73:104–111. https://doi.org/10.1016/j.ceca.2018.04.009
Zhang XH, Wei H, Xia Y, Morad M (2021) 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. Heart Rhythm 18(2):250–260. https://doi.org/10.1016/j.hrthm.2020.09.007
Fernandez-Morales JC, Xia Y, Renzo TJ, Zhang XH, Morad M (2022) Mutation in RyR2-FKBP Binding site alters Ca(2+) signaling modestly but increases “arrhythmogenesis” in human stem cells derived cardiomyocytes. Cell Calcium 101:102500. https://doi.org/10.1016/j.ceca.2021.102500
Lian X, Zhang J, Azarin SM, Zhu K, Hazeltine LB, Bao X et al (2013) Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions. Nat Protoc 8(1):162–175. https://doi.org/10.1038/nprot.2012.150
Yang X, Rodriguez ML, Leonard A, Sun L, Fischer KA, Wang Y et al (2019) Fatty acids enhance the maturation of cardiomyocytes derived from human pluripotent stem cells. Stem Cell Reports 13(4):657–668. https://doi.org/10.1016/j.stemcr.2019.08.013
Pahlavan S, Morad M (2017) Total internal reflectance fluorescence imaging of genetically engineered ryanodine receptor-targeted Ca(2+) probes in rat ventricular myocytes. Cell Calcium 66:98–110. https://doi.org/10.1016/j.ceca.2017.07.003
Acknowledgments
This work was supported by the National Institutes of Health Grants HL147054 and HL153504.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Yamaguchi, N., Zhang, XH., Morad, M. (2022). CRISPR/Cas9 Gene Editing of RYR2 in Human iPSC-Derived Cardiomyocytes to Probe Ca2+ Signaling Aberrancies of CPVT Arrhythmogenesis. In: Ishikawa, K. (eds) Cardiac Gene Therapy. Methods in Molecular Biology, vol 2573. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2707-5_4
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2707-5_4
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2706-8
Online ISBN: 978-1-0716-2707-5
eBook Packages: Springer Protocols