CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of ?2AR in isogenic hPSC-derived cardiomyocytes

Kondrashov, Alexander; Mohd Yusof, Nurul A.N.; Hasan, Alveera; Goulding, Jo�lle; Kodagoda, Thusharika; Hoang, Duc M.; Vo, Nguyen T.N.; Melarangi, Tony; Dolatshad, Nazanin; Gorelik, Julia; Hill, Stephen J.; Harding, Sian E.; Denning, Chris

Abstract

© 2020 During normal- and patho-physiological situations, the behavior of the beta2-adrenoreceptor (β2AR) is influenced by polymorphic variants. The functional impact of such polymorphisms has been suggested from data derived from genetic association studies, in vitro experiments with primary cells, and transgenic overexpression models. However, heterogeneous genetic background and non-physiological transgene expression levels confound interpretation, leading to conflicting mechanistic conclusions. To overcome these limitations, we used CRISPR/Cas9 gene editing technology in human pluripotent stem cells (hPSCs) to create a unique suite of four isogenic homozygous variants at amino acid positions 16(G/R) and 27(G/Q), which reside in the N terminus of the β2AR. By producing cardiomyocytes from these hPSC lines, we determined that at a functional level β2AR signaling dominated over β1AR. Examining changes in beat rates and responses to isoprenaline, Gi coupling, cyclic AMP (cAMP) production, downregulation, and desensitization indicated that responses were often heightened for the GE variant, implying differential dominance of both polymorphic location and amino acid substitution. This finding was corroborated, since GE showed hypersensitivity to doxorubicin-induced cardiotoxicity relative to GQ and RQ variants. Thus, understanding the effect of β2AR polymorphisms on cardiac response to anticancer therapy may provide a route for personalized medicine and facilitate immediate clinical impact. During normal- and patho-physiological situations, the behavior of beta2-adrenoreceptor (β2AR) is determined by its polymorphic variants. A human-based isogenic model system represents a promising tool for systematic analysis of mechanisms of β2AR variant-mediated cellular response under normal and stressed conditions. This allows important subtleties of polymorphisms in β2AR to be unraveled.

Citation

Kondrashov, A., Mohd Yusof, N. A., Hasan, A., Goulding, J., Kodagoda, T., Hoang, D. M., …Denning, C. (2021). CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of β2AR in isogenic hPSC-derived cardiomyocytes. Molecular Therapy - Methods and Clinical Development, 20, 39-53. https://doi.org/10.1016/j.omtm.2020.10.019

• Journal Article Type: Article
• Acceptance Date: Oct 20, 2020
• Online Publication Date: Oct 27, 2020
• Publication Date: Mar 12, 2021
• Deposit Date: Nov 6, 2020
• Publicly Available Date: Nov 6, 2020
• Journal: Molecular Therapy - Methods and Clinical Development
• Print ISSN: 2329-0501
• Electronic ISSN: 2329-0501
• Publisher: Elsevier (Cell Press)
• Peer Reviewed: Peer Reviewed
• Volume: 20
• Pages: 39-53
• DOI: https://doi.org/10.1016/j.omtm.2020.10.019
• Public URL: https://nottingham-repository.worktribe.com/output/5021110
• Publisher URL: https://www.cell.com/molecular-therapy-family/methods/fulltext/S2329-0501(20)30223-0