Distinct functions of cardiac β-adrenergic receptors in the T-tubule vs. outer surface membrane
- Université Paris-Saclay, France
- Université Paris-Saclay, CNRS, Institut Galien Paris-Saclay, France
- Collège de France, INSERM, France
Abstract
β-adrenoceptors (β-ARs) regulate cardiac function during sympathetic nerve stimulation. β-ARs are present in both the cardiac T-tubule (TTM) and outer surface membrane (OSM), but how their location impacts 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 covalently 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 T-tubule 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 of adult rat ventricular myocytes (ARVMs) due to the presence of the extracellular glycocalyx. β-AR activation in OSM with PEG-Iso produced a lower stimulation of [cAMP]i than Iso but a larger stimulation of cytosolic PKA at equivalent levels of [cAMP]I and similar effects on excitation-contraction coupling parameters. However, PEG-Iso produced a much lower stimulation of nuclear cAMP and PKA than Iso. Thus, OSM β-ARs in ARVMs control mainly cytosolic cAMP/PKA pathway and contractility, while TTM β-ARs control mainly nuclear cAMP, PKA and consequent nuclear protein phosphorylation. Size exclusion strategy using ligand PEGylation provides a unique approach to evaluate the respective contribution of T-tubule vs. outer surface membrane proteins in cardiac cells.
Data availability
A dataset named CARDIOPEG has been uploaded to the Dryad website. It contains all the individual experiments used for the figures in the article.
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Distinct functions of cardiac β-adrenergic receptors in the T-tubule vs. outer surface membraneDryad Digital Repository, doi:10.5061/dryad.05qfttf6d.
Article and author information
Author details
Rodolphe Fischmeister
Funding
Agence Nationale de la Recherche (ANR-10-LABX-33)
- Laurence Moine
- Nicolas Tsapis
- Rodolphe Fischmeister
Fondation pour la Recherche Médicale
- Marion Barthe
Agence Nationale de la Recherche (ANR-11-IDEX-0003-01)
- Laurence Moine
- Nicolas Tsapis
- Rodolphe Fischmeister
Agence Nationale de la Recherche (ANR-15-CE14-0014-01)
- Laurence Moine
- Nicolas Tsapis
- Rodolphe Fischmeister
Agence Nationale de la Recherche (ANR-23-CE14-0027-01)
- Laurence Moine
- Nicolas Tsapis
- Rodolphe Fischmeister
Fondation Lefoulon Delalande
- George WP Madders
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal care and experimental procedures complied with the ARRIVE guidelines and conform to the European Community guiding principles in the Care and Use of Animals (Directive 2010/63/EU of the European Parliament), the local Ethics Committee (CREEA Ile-de France Sud) guidelines, and the French decree no. 2013-118 of February 1st, 2013 on the protection of animals used for scientific purposes (JORF no. 0032, February 7th, 2013, p2199, text no. 24). Animal experiments according were carried out according to the European Community guiding principles in the care and use of animals (2010/63/UE), the local Ethics Committee (CREEA Ile-de-France Sud) guidelines and the French decree n{degree sign} 2013-118 on the protection of animals used for scientific purposes.
Copyright
© 2025, Madders et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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Distinct functions of cardiac β-adrenergic receptors in the T-tubule vs. outer surface membrane
eLife 14:e84243.
https://doi.org/10.7554/eLife.84243
