Abstract 16646: Inositol 1,4,5-Trisphosphate Receptor Regulation in Atrial Myocyte Microdomains

Abstract

Introduction: InsP 3 receptor type 2 (InsP 3 R2) is up-regulated in patients with atrial fibrillation (AF) and InsP 3 -induced Ca release (IICR) is linked to an increase in premature atrial contractions (PACs). Nevertheless, knowledge of the physiological and pathophysiological relevance and regulation of InsP 3 Rs in the atria is limited. Hypothesis: We hypothesized that InsP 3 R and NADPH oxidase 2 (NOX2) form a functional signaling domain where NOX2 derived reactive oxygen species (ROS) regulate InsP 3 R agonist affinity and contractile strength. Method: Atrial myocytes (AM) were isolated from wild type and NOX2 (gp91 phox-/- ) deficient mice. Changes in cytoplasmic Ca concentration [Ca] i ; fluo-4/AM) or ROS (2’,7’-dichlorofluorescein, DCF) were monitored by confocal microscopy. Results: We demonstrate for the first time in AMs that agonist angiotensin II (AngII) induced IICR and PACs depend on ROS production in a signaling domain of InsP 3 R and NOX2. Superfusion of AMs with AngII (1 μM) significantly increased diastolic [Ca] i (ΔF/F 0 , Ctrl 20’ : 1.0±0.01, AngII 20’ : 1.2±0.03; n=7; p<0.05), the field stimulation induced Ca transient amplitude (ΔF/F 0 , Ctrl: 2.0±0.17, AngII: 2.39±0.22, n=7; p<0.05), and spontaneous Ca releases resulting in PACs (Ctrl: 0 s -1 , AngII: 0.15±0.05 s -1 ; n=7; p<0.05). These effects, were suppressed by InsP 3 R2 blocker 2-aminoethoxydiphenyl-borate (2APB; 1 μM)) and phopholipase C inhibitor, U73122 (10 μM), demonstrating their dependence on IICR. Simultaneously, AngII induced an increase in ROS production that was sensitive to the NOX2 specific inhibitor gp91ds-tat (1 μM). In AMs deficient for NOX2 (gp91 phox-/- ) AngII failed to increase [Ca] i and to induce PACs indicating a sensitization of InsP 3 R by NOX2/ROS. In saponin (0.005%) permeabilized AMs InsP 3 (5 μM) induced spatially defined Ca release events (sparks) that increased in frequency in presence of exogenous ROS (tert-Butyl hydroperoxide: tBHP: 5 μM; InsP 3 : 9.65±1.44 sparks*s - 1 (100 μm -1 ); InsP3 + tBHP: 10.77±1.5 sparks*s -1 (100 μm -1 ), n=5, p<0.05). The combined effect of InsP 3 + tBHP was entirely suppressed by 2-APB suggesting a ROS dependent sensitization of the ryanodine receptor through IICR. Conclusion: Our data show a novel mechanism of InsP 3 R regulation in the atrial muscle that consisting of a functional signaling domain where InsP 3 R and NOX2 closely interact. This mechanism could have relevance in a stretch dependent increase in atrial contractile force.