Article Text

Download PDFPDF

BS49 Human embryonic stem cell derived cardiomyocytes express functional receptors for the cardiovascular peptide apelin
Free
  1. Robyn Macrae,
  2. William G. Bernard,
  3. Rhoda E. Kuc,
  4. Maria T. Colzani,
  5. Thomas Williams,
  6. Duuamene Nyimanu,
  7. Janet Maguire,
  8. Sanjay Sinha,
  9. Anthony P. Davenport
  1. University of Cambridge

Abstract

Introduction The apelin receptor is expressed throughout the cardiovascular system, including in cardiomyocytes. Receptor activation by either of its endogenous peptide ligands, apelin or Elabela, has a positive inotropic effect and promotes vasodilatation. Human embryonic stem cell (hESC)-derived cells have the potential for use in translational research to investigate cellular signalling, disease pathogenesis and potential novel treatments. Our aim was to determine if hESC-derived cardiomyocytes express apelin receptor protein and to quantify receptor density to ascertain if this cell type can be used as a phenotypic model for human diseases associated with apelin receptor mutations.

Methods H9 hESCs were cultured to induce differentiation to beating cardiomyocytes. Saturation radioligand binding experiments were performed using [Glp65,Nle75,Tyr77][125I]apelin-13 and [Pyr1]apelin-13 to define non-specific binding. Bound radioactivity was counted and data analysed using iterative curve fitting programs to obtain values of receptor affinity (KD) and density (BMAX). Immunocytochemistry was carried out using anti-apelin receptor or anti-cardiac cell marker antibodies.

Results Previous work demonstrated expression of the apelin receptor in hESC-derived cardiomyocytes at the gene level by qRT-PCR at similar levels to adult cells (figure 1A). Here, radioligand binding studies have confirmed receptor protein expression in beating hESC-derived cardiomyocytes. Binding was saturable and [125I]apelin-13 bound with sub-nanomolar affinity (0.12 nM) and receptor density found to be 21 fmol/mg (figure 1B), comparable to that found in human adult heart. Hill slope was close to 1 consistent with a single binding site for apelin in these cells. Furthermore, beating hESC-derived cardiomyocytes stained positive for apelin receptor, in addition to the standard cardiac markers including cardiac troponin T (figure 2).

Abstract BS49 Figure 1 (A) Relative expression of apelin receptor (APLNR) gene in hESC-derived cardiomyocytes(CM). Expression is displayed relative to 18S. (B) Saturation binding curve for beating hESC-derived cardiomyocytes when incubated with increasing concentrations of [125I]apelin-13

Abstract BS49 Figure 2 Representative image of anti-apelin receptor (green) staining in beating hESC-derived cardiomyocytes. Blue = DAPI, scale bar = 100 μm

Conclusion These data importantly confirm that hESC-derived cardiomyocytes express apelin receptor protein at similar levels to adult human heart. Apelin receptor mutations have been identified in the 100,000 Genomes Bridge Project that are associated with rare diseases, including pulmonary arterial hypertension. Our ongoing experiments aim to pharmacologically characterise the apelinergic signalling pathway in the beating hESC-derived cardiomyocytes and we propose to generate hESC-derived phenotypic models by introducing selected apelin receptor mutations via CRISPR/Cas-9 gene editing.

Conflict of interest None

  • Stem cells
  • Apelin
  • Cardiomyocytes

Statistics from Altmetric.com

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.