CRISPR/Cas9 mediated generation and analysis of N-terminus polymorphic models of beta2-adrenoreceptor in isogenic hPSC derived cardiomyocytes.

Abstract During normal- and patho-physiological situations, the behaviour of the beta2-adrenoreceptor (β2AR) is influenced by polymorphic variants. The functional impact of such polymorphisms have 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 signalling dominated over β1AR . Examining changes in beat rates and responses to isoprenaline, Gi coupling, cAMP production, downregulation and desensitisation 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 personalised medicine and facilitate immediate clinical impact.