Functional Microdomain of Adenylyl Cyclase Isoform 1 Contributes to Sinoatrial Node Automaticity via β-Adrenergic Receptor Pathway

Abstract

The sinoatrial node (SAN) is the primary pacemaker of the heart and is tightly regulated by β-adrenergic receptor (β-AR) signaling. Adenylyl cyclase (AC) is a key enzyme in the β-AR pathway that catalyzes the production of cAMP. We hypothesize that compartmentalization of different isoforms of ACs with the membrane and Ca2+ clocks are critical for the function of SAN cells (SANC). Immunofluorescence confocal microscopy, stimulated emission depletion microscopic imaging, and proximity ligation assay (PLA) were used to examine compartmentalization of ACI and caveolin-3 (Cav3), HCN4, or Cav1.2. Spontaneous action potentials, pacemaker and Ca2+ currents, as well as local Ca2+ release were recorded. cAMP-FRET biosensors targeting plasma membrane, sarcoplasmic reticulum, and cytosol were expressed in cultured SANC isolated from ACI-/-and wild type (WT) mice to examine AC functional microdomains in the SAN. We demonstrate that ACI is the most abundant isoform in the SAN. ACI-/-mice exhibited SAN dysfunction after β-AR stimulation. To elucidate the mechanistic underpinnings, we examined subcellular co-localization of ACI to key proteins that contribute to functional integrity of the SAN. We observed robust PLA signals of SANCs co-labeled for ACI with Cav3, HCN4, or Cav1.2, Functionally, there were significant differences in action potential firing frequencies, Ca2+ and pacemaker currents between SANCs isolated from ACI-/-and WT mice. Novel cAMP biosensors showed a blunted response in local cAMP generation during β-AR stimulation in ACI-/-SANC. ACI is the predominant isoform in the SAN and co-localizes with Cav3 and HCN4 channels. Compartmentalization of ACI and HCN4 plays critical roles in the regulation of the “membrane clock” via β-AR pathway. Since ACI is Ca2+-activated, it may provide the molecular link between Ca2+ and membrane clocks of the SAN.