Relevance Of Insp3 Receptor Ros Regulation In Atrial Myocytes

In atrial tissue InsP3 induced InsP3 receptor type 2 (InsP3R2) mediated Ca release (IICR) has been linked to positive inotropy as well as to increased arrhythmic Ca- dependent after-depolarizations. InsP3 production in atrial myocytes is regulated through Gq protein coupled receptors like α1-adrenergic, endothelin-1 or Angiotensin II (AngII) receptors and concomitant stimulation of PLC. Here we tested the hypothesis that post-translational modifications of InsP3Rs in cardiomyocytes have significant consequences for cardiac excitation-contraction. Superfusion of field stimulated mouse atrial myocytes with AngII (1μM) induced a significant increase in diastolic [Ca], the Ca transient amplitude as well as arrhythmic after-depolarizations. This increase in [Ca]i was suppressed by treatment of the cells with the InsP3R2 blocker 2-Aminoethoxydiphenyl borate (2APB) as well as by the phopholipase C inhibitor U73122 (10μM) supporting the involvement of IICR. Simultaneously to changes in [Ca]i, AngII promotes the stimulation of NADPH oxidase 2 (NOX2) dependent ROS production. To determine if ROS contributes to AngII induced changes in [Ca]i the experiments were repeated in mice deficient for the expression of NOX2 (gp91phox-/-, p47phox-/-). In NOX2 KO myocyes, AngII remained without effect on [Ca]i supporting the notion that IICR mediated increase in [Ca]i depends on the presence of NOX2/ROS. ROS-dependent S-glutathionylation of InsP3R2 increases their affinity to InsP3 whereas it increases the ryanodine receptors (RYR) open probability. To determine the role of S-Glutathionlylation, atrial myocytes were superfused with Diamide (100μM). Diamide, comparable to AngII, induced an increase in basal and Ca transient amplitude leading to arrhythmic events. The change was reversible upon superfusion with the antioxidant DTT (1 mM) and preventable by pre-treatment with 2APB. The data support that ROS-dependent regulation of IICR plays a prominent role in the regulation atrial [Ca]i handling properties and is a sensitive readout for NOX2/ROS production.