Fluorescence spectroscopy of low-level endogenous β-Adrenergic Receptor expression at the plasma membrane of differentiating human iPSC-derived cardiomyocytes

Abstract

The potential of human induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking the adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the {beta}1- and {beta}2-adrenergic receptors ({beta}1/2-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetic of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report that {beta}2-ARs, as opposed to {beta}1-ARs, specifically segregate to the T-Tubular system of adult CMs.