Dynamic mass redistribution analysis of endogenous b-adrenergic receptor signaling in neonatal rat cardiacfibroblasts.pdf

Label-free systems for the agnostic assessment of cellular responses to receptorstimulation have been shown to provide a sensitive method to dissect receptorsignaling.b-adenergic receptors (bAR) are important regulators of normal andpathologic cardiac function and are expressed in cardiomyocytes as well as car-diac fibroblasts, where relatively fewer studies have explored their signalingresponses. Using label-free whole cell dynamic mass redistribution (DMR)assays we investigated the response patterns to stimulation of endogenousbARin primary neonatal rat cardiac fibroblasts (NRCF). The EPIC-BT by Corningwas used to measure DMR responses in primary isolated NRCF treated withvariousbAR and EGFR ligands. Additional molecular assays for cAMP genera-tion and receptor internalization responses were used to correlate the DMRfindings with establishedbAR signaling pathways. Catecholamine stimulation ofNRCF induced a concentration-dependent negative DMR deflection that wascompetitively blocked bybAR blockade and non-competitively blocked by irre-versible uncoupling of Gs proteins. Subtype-selectivebAR ligand profilingrevealed a dominant role forb2AR in mediating the DMR responses, consistentwith the relative expression levels ofb2AR andb1AR in NRCF.bAR-mediatedcAMP generation profiles revealed similar kinetics to DMR responses, each ofwhich were enhanced via inhibition of cAMP degradation, as well as dynamin-mediated receptor internalization. Finally, G protein-independentbAR signalingthrough epidermal growth factor receptor (EGFR) was assessed, revealing asmaller but significant contribution of this pathway to the DMR response tobAR stimulation. Measurement of DMR responses in primary cardiac fibro-blasts provides a sensitive readout for investigating endogenousbAR signalingvia both G protein-dependent and–independent pathways