Abstract
Adult rat ventricular myocytes (ARVMs) membrane contains invaginations (transverse tubule membrane, TTM). TTM is continuous with the outer surface membrane (OSM) to allow rapid electrical coupling during the action potential. β-adrenergic receptors (βAR) are located in both OSM and TTM, but at different densities. TT provides the development of dyadic junctions with the sarcoplasmic reticulum, which is essential for excitation-contraction coupling (ECC). Yet, how β-AR compartmentation modulates ECC remains unknown. To selectively activate βARs in OSM and not in TTM using a steric hindrance approach. We covalently linked a 5 kDa PolyEthylene-Glycol (PEG) on βAR agonist (Isoprenaline, Iso) or antagonist (Alprenolol, Alp) to increase the size of the βAR ligand and prevent its access to TTM. Chemical grafting and PEG-ligand purification were optimized. A fluorescent PEG (PEG-FITC) was used to visualize its distribution in ARVMs. Pharmacological characterization of the PEG-ligands was performed using radioligand binding assays on β1 or β2AR, and cAMP measurement by FRET in ARVMs. The effects of PEG-ligands on ECC were examined in ARVMs using the Ionoptix system. (1) PEG-FITC does not penetrate in the TT network. (2) The chemical grafting was validated. (3) PEG-ligands bind to β1 and β2AR with Ki values of, respectively: 1300 and 430 μM ( n = 5) for PEG-Iso, and 20.0 and 5.6 μM ( n = 5) for PEG-Alp. (4) PEG-Iso produces a dose-dependent increase in cytosolic cAMP. The maximal response is comparable to free Iso. (5) PEG-Alp antagonizes the cytosolic cAMP response to free Iso, with a maximal effect close to full inhibition. (6) Activation of βAR at the OSM, using PEG-Iso, has smaller impact on ECC than global activation using free Iso. Our results indicate that (1) activation of βARs at the OSM is sufficient to produce maximal elevation of cytosolic cAMP, but (2) βARs in the TTM are more efficiently coupled to contractility and Ca2+ regulation.
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