Differential Roles of Calcium-Activated and Calcium-Inhibited Adenylyl Cyclase Isoforms in the Regulation of SAN Automaticity

Abstract

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 from ATP, leading to the activation of a cascade of downstream effectors. There are 10 known isoforms of AC, but their relative expression and distribution vary considerably among different regions of the heart. Additionally, a knowledge gap exists regarding their contribution to SAN automaticity. We hypothesize that Ca2+-activated AC isoforms play important regulatory roles in SAN automaticity. To determine regional distribution of AC isoforms in the heart, single-cell RT-PCR was performed. Global knockout (KO) of AC isoforms and WT C57Bl/6 mice were utilized to assess the role of AC in the SAN at basal and after β-AR stimulation with isoproterenol (ISO). Cardiac structure and function were assessed by echocardiography as well as hemodynamic measurements. Ambulatory electrocardiography was used to determine in vivo SAN function during basal condition, after β-AR stimulation, and after autonomic nervous system blockade. Pacemaker and Ca2+ currents, as well as local Ca2+ release, were performed in isolated SAN cells using conventional patch-clamp techniques and confocal microscopy. cAMP biosensors were used to directly evaluate compartmentalization of the different isoforms. Indeed, we found distinct regulation of SAN automaticity by different isoforms of ACs. Differential compartmentalization of AC isoforms with the Ca2+vs. membrane clocks further validated these findings. Localization and contribution to SAN automaticity vary with different isoforms of ACs due to their distinct compartmentalization with the Ca2+vs. membrane clocks. The findings have broad therapeutic ramifications. Embedded in these findings are novel insights that can be exploited in developing successful therapeutics for SAN dysfunction.