FRET Measurements of cAMP Dynamics in HL1 Cells Support the Key Role of Constitutive AC Activity in Cardiac Pacemaking

Abstract

Spontaneously beating HL1 cardiac cells have evolved as a very useful tool to study regulation of pacemaking cardiac activity because of the ability to knock down specific molecules without generating KO mice in vivo. HL1 cells, however, have not been fully characterized and their pacemaker mechanisms may differ from those in cardiac pacemaking cells derived from adult heart. Basal constitutive adenylate cyclase activity is an obligate factor for normal spontaneous firing in adult sinoatrial nodal pacemaker cells.We expressed FRET sensors in spontaneously beating HL1 cardiac cells to measure cAMP concentration. The PDE inhibitor IBMX (100 μM) increased the basal concentration of cAMP more than twofold and increased beating frequency by 70%. AC1 knockdown with siRNA completely inhibited the IBMX-induced increase in cAMP production in cytoplasm and plasma membrane, and decreased the beating frequency of HL1 cells. Pharmacological inhibition of both plasma membrane AC (2′-5’ dideoxyadenosine) and cytoplasmic AC (KH7) selective blockers completely abolished the IBMX induced increase of cAMP concentration and resulted in complete cessation of spontaneous beating in HL1cardiac cells. Thus, as in adult sinoatrial nodal pacemaker cells, basal AC activity is required for the spontaneous beating of HL1 cardiac cells.