Inositol 1,4,5-trisphosphate receptor - reactive oxygen signaling domain regulates excitation-contraction coupling in atrial myocytes

The inositol 1,4,5-trisphosphate receptor (InsP₃R) is up-regulated in patients with atrial fibrillation (AF) and InsP₃-induced Ca²⁺ release (IICR) is linked to pro-arrhythmic spontaneous Ca²⁺ release events. Nevertheless, knowledge of the physiological relevance and regulation of InsP₃Rs in atrial muscle is still limited. We hypothesize that InsP₃R and NADPH oxidase 2 (NOX2) form a functional signaling domain where NOX2 derived reactive oxygen species (ROS) regulate InsP₃R agonist affinity and thereby Ca²⁺ release. To quantitate the contribution of IICR to atrial excitation-contraction coupling (ECC) atrial myocytes (AMs) were isolated from wild type and NOX2 deficient (Nox2^-/-) mice and changes in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i; fluo-4/AM, indo-1) or ROS (2',7'-dichlorofluorescein, DCF) were monitored by fluorescence microscopy. Superfusion of AMs with Angiotensin II (AngII: 1 μmol/L) significantly increased diastolic [Ca²⁺]_i (F/F₀, Ctrl: 1.00 ± 0.01, AngII: 1.20 ± 0.03; n = 7; p < 0.05), the field stimulation induced Ca²⁺ transient (CaT) amplitude (ΔF/F₀, Ctrl: 2.00 ± 0.17, AngII: 2.39 ± 0.22, n = 7; p < 0.05), and let to the occurrence of spontaneous increases in [Ca²⁺]_i. These changes in [Ca²⁺]_i were suppressed by the InsP₃R blocker 2-aminoethoxydiphenyl-borate (2-APB; 1 μmol/L). Concomitantly, AngII induced an increase in ROS production that was sensitive to the NOX2 specific inhibitor gp91ds-tat (1 μmol/L). In NOX2^-/- AMs, AngII failed to increase diastolic [Ca²⁺]_i, CaT amplitude, and the frequency of spontaneous Ca²⁺ increases. Furthermore, the enhancement of CaTs by exposure to membrane permeant InsP₃ was abolished by NOX inhibition with apocynin (1 μM). AngII induced IICR in Nox2^-/- AMs could be restored by addition of exogenous ROS (tert-butyl hydroperoxide, tBHP: 5 μmol/L). In saponin permeabilized AMs InsP₃ (5 μmol/L) induced Ca²⁺ sparks that increased in frequency in the presence of ROS (InsP₃: 9.65 ± 1.44 sparks*s^-1*(100μm)^-1; InsP₃ + tBHP: 10.77 ± 1.5 sparks*s^-1*(100μm)^-1; n = 5; p < 0.05). The combined effect of InsP₃ + tBHP was entirely suppressed by 2-APB and Xestospongine C (XeC). Changes in IICR due to InsP₃R glutathionylation induced by diamide could be reversed by the reducing agent dithiothreitol (DTT: 1 mmol/L) and prevented by pretreatment with 2-APB, supporting that the ROS-dependent post-translational modification of the InsP₃R plays a role in the regulation of ECC. Our data demonstrate that in AMs the InsP₃R is under dual control of agonist induced InsP₃ and ROS formation and suggest that InsP₃ and NOX2-derived ROS co-regulate atrial IICR and ECC in a defined InsP₃R/NOX2 signaling domain.