title: Dynamic mass redistribution analysis of endogenous β-adrenergic receptor signaling in neonatal rat cardiac fibroblasts.
abstract: Label-free systems for the agnostic assessment of cellular responses to receptor stimulation have been shown to provide a sensitive method to dissect receptor signaling. β-adenergic receptors (βAR) are important regulators of normal and pathologic cardiac function and are expressed in cardiomyocytes as well as cardiac fibroblasts, where relatively fewer studies have explored their signaling responses. Using label-free whole cell dynamic mass redistribution (DMR) assays we investigated the response patterns to stimulation of endogenous βAR in primary neonatal rat cardiac fibroblasts (NRCF). Catecholamine stimulation of the cells induced a negative DMR deflection resulting in a concentration-dependent pharmacological response that was competitively blocked by βAR blockade and non-competitively blocked by irreversible uncoupling of Gs proteins. Pharmacological profiling of subtype-selective βAR agonists and antagonists revealed a dominant role of β2AR in mediating the DMR responses, consistent with the relative expression levels of β2AR and β1AR in NRCF. Additionally, βAR-mediated cAMP generation was assessed via a fluorescence biosensor, revealing similar kinetics between DMR responses and cAMP generation. As such, βAR-dependent DMR responses were enhanced via inhibition of cAMP degradation, as well as dynamin-mediated receptor internalization. Finally, we assessed G protein-independent βAR signaling through epidermal growth factor receptor (EGFR). While inhibition of EGFR reduced the DMR response to βAR stimulation, our results demonstrate that G protein-dependent signaling produces a majority of the biological response to βAR stimulation in NRCF. Altogether, measurement of DMR responses in primary cardiac fibroblasts provides a sensitive readout for investigating endogenous βAR signaling via both G protein-dependent and -independent pathways.
authors.affiliation: Center for Translational Medicine (R.L.C., L.A.G., J.E.Y., A.A.R., M.W., J.I., W.J.K. and D.G.T.) and Department of Pharmacology (W.J.K. and D.G.T.), Temple University School of Medicine, and Moulder Center for Drug Discovery Research and Temple University School of Pharmacy (M.A.J.), Philadelphia, PA 19140, USA.
authors.forename: Rhonda L
authors.initials: RL
authors.lastname: Carter
authors.affiliation: Center for Translational Medicine (R.L.C., L.A.G., J.E.Y., A.A.R., M.W., J.I., W.J.K. and D.G.T.) and Department of Pharmacology (W.J.K. and D.G.T.), Temple University School of Medicine, and Moulder Center for Drug Discovery Research and Temple University School of Pharmacy (M.A.J.), Philadelphia, PA 19140, USA.
authors.forename: Laurel A
authors.initials: LA
authors.lastname: Grisanti
authors.affiliation: Center for Translational Medicine (R.L.C., L.A.G., J.E.Y., A.A.R., M.W., J.I., W.J.K. and D.G.T.) and Department of Pharmacology (W.J.K. and D.G.T.), Temple University School of Medicine, and Moulder Center for Drug Discovery Research and Temple University School of Pharmacy (M.A.J.), Philadelphia, PA 19140, USA.
authors.forename: Justine E
authors.initials: JE
authors.lastname: Yu
authors.affiliation: Center for Translational Medicine (R.L.C., L.A.G., J.E.Y., A.A.R., M.W., J.I., W.J.K. and D.G.T.) and Department of Pharmacology (W.J.K. and D.G.T.), Temple University School of Medicine, and Moulder Center for Drug Discovery Research and Temple University School of Pharmacy (M.A.J.), Philadelphia, PA 19140, USA.
authors.forename: Ashley A
authors.initials: AA
authors.lastname: Repas
authors.affiliation: Center for Translational Medicine (R.L.C., L.A.G., J.E.Y., A.A.R., M.W., J.I., W.J.K. and D.G.T.) and Department of Pharmacology (W.J.K. and D.G.T.), Temple University School of Medicine, and Moulder Center for Drug Discovery Research and Temple University School of Pharmacy (M.A.J.), Philadelphia, PA 19140, USA.
authors.forename: Meryl
authors.initials: M
authors.lastname: Woodall
authors.affiliation: Center for Translational Medicine (R.L.C., L.A.G., J.E.Y., A.A.R., M.W., J.I., W.J.K. and D.G.T.) and Department of Pharmacology (W.J.K. and D.G.T.), Temple University School of Medicine, and Moulder Center for Drug Discovery Research and Temple University School of Pharmacy (M.A.J.), Philadelphia, PA 19140, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Ibetti
authors.affiliation: Center for Translational Medicine (R.L.C., L.A.G., J.E.Y., A.A.R., M.W., J.I., W.J.K. and D.G.T.) and Department of Pharmacology (W.J.K. and D.G.T.), Temple University School of Medicine, and Moulder Center for Drug Discovery Research and Temple University School of Pharmacy (M.A.J.), Philadelphia, PA 19140, USA.
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Koch
authors.affiliation: Center for Translational Medicine (R.L.C., L.A.G., J.E.Y., A.A.R., M.W., J.I., W.J.K. and D.G.T.) and Department of Pharmacology (W.J.K. and D.G.T.), Temple University School of Medicine, and Moulder Center for Drug Discovery Research and Temple University School of Pharmacy (M.A.J.), Philadelphia, PA 19140, USA.
authors.forename: Marlene A
authors.initials: MA
authors.lastname: Jacobson
authors.affiliation: Center for Translational Medicine (R.L.C., L.A.G., J.E.Y., A.A.R., M.W., J.I., W.J.K. and D.G.T.) and Department of Pharmacology (W.J.K. and D.G.T.), Temple University School of Medicine, and Moulder Center for Drug Discovery Research and Temple University School of Pharmacy (M.A.J.), Philadelphia, PA 19140, USA.
authors.forename: Douglas G
authors.initials: DG
authors.lastname: Tilley
keywords: cardiac fibroblast; dynamic mass redistribution; epidermal growth factor receptor; fluorescence resonance energy transfer; β-adrenergic receptor