Functional β-adrenergic receptor signalling on nuclear membranes in adult rat and mouse ventricular cardiomyocytes

Objective: We sought to determine if different beta-adrenergic receptor (beta AR) subtypes, and their associated signalling machinery, are functionally localized to nuclear membranes. Methods: Employing enriched nuclear preparations, we assayed the specific presence of beta AR by measuring I-125-cyanopindolol (CYP) binding, Western blotting, confocal microscopy and functional assays. Results: Western blots of rat heart nuclear fractions and confocal immunofluorescent analysis of adult rat and mouse ventricular cardiornyocytes displayed the presence of beta(1)AR and beta(3)AR but, surprisingly, not the beta(2)AR on nuclear membranes. Nuclear localization of downstream signalling partners Gs, Gi and adenylyl cyclases II and V/VI was also demonstrated. The functional relevance of nuclear beta AR was shown by receptor-mediated stimulation of adenylyl cyclase activity by isoproterenol but not the beta(3)AR-selective agonist CL 316243 in enriched nuclear preparations. We also examined the effect of subtype-selective ligands on the initiation of RNA synthesis in isolated nuclei. Both isoproterenol and another MR-selective agonist, BRL 37344, increased RNA synthesis which was inhibited by pertussis toxin (PTX). Neither a beta(1)AR-selective agonist, xamoterol, nor a beta(2)AR-selective agonist, procaterol, was able to stimulate transcription. However, both CGP 20712A and ICI 118,551 blocked isoproterenol-mediated effects to varying extents. PTX treatment also revealed that nuclear beta AR may be coupled to other signalling pathways in addition to Gi, as stimulation under these conditions reduced initiation of transcription below basal levels. Conclusion: These results highlight differential subcellular localization for beta AR subtypes and indicate that beta AR may have specific roles in regulating nuclear function in cardiomyocytes. (c) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.