Abstract 318: Dynamic Mass Redistribution Analysis Of Endogenous β-adrenergic Receptor Signaling In Cardiac Cells

Abstract

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 the biological responses to βAR stimulation. We used label-free dynamic mass redistribution (DMR) to elucidate the biological response to stimulation of endogenous βAR in primary rat neonatal cardiac fibroblasts, and to begin to explore the pathways responsible for these effects. Isoproterenol (ISO, non-selective for β1/β2), salbutamol (Sal, β2-selective) and dobutamine (Dob, β1-selective) concentration-dependently induced increases in DMR response, with the Dob-induced response significantly less than either ISO- or Sal-induced DMR, consistent with the reduced expression of β1AR vs β2AR in cardiac fibroblasts. Addition of propranolol (Prop) competitively blocked the DMR effect induced by all 3 agonists, while the addition of cholera toxin (CTX) non-competitively blocked the effects of each agonist. Overall, the CTX-sensitive DMR effect accounted for approximately 75% of the response, while 25% of the biological response was CTX-insensitive. To confirm that the CTX-sensitive DMR response was mediated by downstream Gαs protein-dependent signaling, cAMP generation was assessed via FRET where ISO produced a similar concentration-effect curve and EC50 value as attained with DMR. All of the cAMP generation response was ablated by both Prop and CTX, indicating that the CTX-sensitive acute DMR effect to βAR stimulation is directly related to cAMP generation. Since ~25% of the DMR effect was not CTX-sensitive, other Gαs protein-independent signaling pathways are presumably activated in response to βAR stimulation in cardiac fibroblasts. To assess this possibility, EGFR internalization was quantitated as an indicator of βAR-mediated EGFR transactivation. EGFR internalization increased in response to ISO stimulation in a concentration-dependent manner, with a similar EC50 value as attained in the DMR assay. Defining the relative contributions of different βAR pathways in cardiac fibroblasts will help establish the importance of these pathways as potential therapeutic targets in the heart.