Abstract 16982: Adenylyl Cyclase Isoform 1 Plays a Critical Role in Beta-Adrenergic Response of Pacemaking Cells in Sinoatrial Node

Abstract

Introduction: Sinoatrial node (SAN), the primary pacemaker region of the heart, is the main determinant of heart rate, which is tightly regulated by β-adrenergic receptor (β-AR) signaling. Adenylyl cyclase (AC) is a key enzyme in the β-AR signaling pathway. There are 10 known AC isoforms but it is unclear which AC isoforms are predominant in the SAN. Furthermore, their functional roles in regulating the pacemaking activity of the heart is not well understood. Hypothesis: We hypothesize that distinct AC isoforms are preferentially expressed in SAN and serve specialized function compared to atrial and ventricular myocytes. Methods: Regional distribution of AC isoforms in the heart was determined using single-cell RT-PCR and single molecule fluorescence in situ hybridization. Transgenic mice expressing GCaMP8 under HCN4 promoter was used to identify SAN cells. To determine the role of AC isoforms in the SAN, CRISPR/Cas9 gene editing was used to generate SAN specific knockout (KO) mice. Electrocardiogram was performed to determine heart rate during basal condition and after β-AR stimulation with isoproterenol (ISO). Analyses of Ca 2+ and pacemaking currents as well as local Ca 2+ release were performed in single isolated SAN cells from the KO mice with and without β-AR stimulation. Results: Single-cell RT-PCR showed that in the SAN, AC I is the most abundant isoform, followed by AC VI , AC VII , AC III and AC IV . In contrast, AC VI was the most abundant isoform in both atria and ventricles. CRISPR/Cas9-mediated gene silencing of AC I resulted in a decrease in heart rate with blunted response to ISO (Figure 1). In contrast, AC V /AC VI double KO mice exhibited similar heart rate responses to ISO compared to WT mice, suggesting that AC V /AC VI isoforms do not play critical roles in controlling the SAN firing frequency. Conclusions: Our study suggests AC I plays a critical role in β-AR response of pacemaking cells in SAN and may offer unique therapeutic opportunities to directly modify pacemaking cells without interfering with ventricular myocytes.