Distinct functions of cardiac β-adrenergic receptors in the T-tubulevs.outer surface membrane

β-adrenoceptors (β-ARs) regulate cardiac function during sympathetic nerve stimulation. β-ARs are present in both cardiac T-tubule (TTM) and outer surface membrane (OSM), but how their location impacts on their function is unknown. Here, we developed a technology based on size exclusion to explore the function of β-ARs located in the OSM. We synthetized a PEG-Iso molecule by covalent linking isoprenaline (Iso) to a 5000 Da PolyEthylene-Glycol (PEG) chain to increase the size of the β-AR agonist and prevent it from accessing the TT network. The affinity of PEG-Iso and Iso on β₁- and β₂-ARs was measured using radioligand binding. Molecular dynamics simulation was used to assess PEG-Iso conformation and visualise the accessibility of the Iso moiety to water. Using confocal microscopy, we show that PEGylation constrains molecules outside the T-tubule network due to the presence of the extracellular matrix. β-AR activation in OSM with PEG-Iso produced a lower stimulation of [cAMP]_ithan Iso but a larger stimulation of cytosolic PKA at equivalent levels of [cAMP]_Iand similar effects on excitation-contraction coupling parameters. However, PEG-Iso produced a much lower stimulation of nuclear PKA than Iso. Thus, OSM β-ARs control mainly cytosolic cAMP/PKA pathway and contractility, while TTM β-ARs control mainly nuclear PKA and nuclear protein phosphorylation. Size exclusion strategy using ligand PEGylation provides a unique approach to evaluate the respective contribution of T-tubulevs.outer surface membrane proteins in cardiac cells.