Fig. 8: Using base editing to introduce the R/H168^4.64 substitution into endogenously expressed apelin receptor reduces binding and induces phenotypic dysfunction in a human hESC-CM model.

a Schematic showing experimental pipeline. Cytosine base editing technology was used to introduce the R/H1684.64 substitution into endogenously expressed apelin receptor in human embryonic stem cells (hESCs) originally harvested from the inner cell mass of a human blastocyst, or into differentiated cardiomyocytes (hESC-CMs). Cells were characterised for R/H168^4.64 apelin receptor mRNA expression, protein expression, and radioligand binding. Functional consequences of the R/H168^4.64 substitution were also assessed. Created in BioRender. Davenport, A. (2024) https://BioRender.com/e87y890. b Relative apelin receptor gene (APLNR) expression in wild-type (WT) or R/H168^4.64 variant hESC (solid bars) and hESC-CM (slashed bars) lines. Data expressed as n = 2 for WT hESC, n = 4 (mean ± SD) for WT hESC-CMs, and n = 8 (mean ± SD), for R/H168^4.64 hESC and hESC-CMs. c Representative confocal fluorescent images (n = 4 independent experiments) of WT and R/H1684.64 hESC-CMs treated with anti-apelin receptor antibody (visualised in green) and nuclear marker (visualised in blue) (upper panels) or 300 nM apelin647 fluorescent ligand (visualised in red) (lower panels). Control WT hESC-CMs (upper) were treated with secondary antibody in the absence of the primary, or (lower) in the presence of 10 µM unlabelled [Pyr¹]apelin-13. Scale bars indicate 50 µm. d Saturation radioligand binding curves showing specific binding of [¹²⁵I]-apelin-13 in hESC (upper) or hESC-CM (lower) membrane preparations. Non-specific binding was determined in the presence of 10 µM [Pyr¹]apelin-13. Data expressed as mean ± SD, n = 3 independent experiments. e Functional consequences of the R/H1684.64 substitution in hESC-CMs. Bar charts show differentiation efficiency measured by % hESC-CMs staining positive for troponin T (TnT) (left), apelin peptide concentrations secreted from hESC-CMs as determined by ELISA (middle), and time-to-peak (TTP) voltage per unit time (ms) (right). For all panels here, data are expressed as mean ± SD, n ≥ 3 independent experiments. Statistical significance was determined using a one-way ANOVA, with Tukey’s correction for multiple comparisons looking for differences between wild-type (WT) and variant (R/H168^4.64) apelin receptor. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.