NADPH oxidase 2 mediates angiotensin II-dependent cellular arrhythmias via PKA and CaMKII

Rationale: Angiotensin II (Ang II) signaling has been implicated in cardiac arrhythmogenesis, which involves induction of reactive oxygen species (ROS). It was shown that Ang II can activate Ca/Calmodulin kinase II (CaMKII) by oxidation via a NADPH oxidase 2 (NOX2)-dependent pathway leading to increased arrhythmic afterdepolarizations. Interestingly, cAMP-dependent protein kinase A (PI(A) which regulates similar targets as CaMKII has recently been shown to be redox-sensitive as well. Objective: This study aims to investigate the distinct molecular mechanisms underlying Aug II-related cardiac arrhythmias with an emphasis on the individual contribution of PICA vs. CaMKII. Methods and results: Isolated ventricular cardiac myocytes from rats and mice were used. Ang II exposure resulted in increased NOX2-dependent ROS generation assessed by expression of redox-sensitive GFP and in myocytes loaded with ROS indicator MitoSOX. Whole cell patch clamp measurements showed that Ang II significantly increased peak Ca and Na current (I-Ca and I-Na) possibly by enhancing steady-state activation of I-Ca and I-Na These effects were absent in myocytes lacking functional NOX2 (gp91phox(-/-)). In parallel experiments using PKA inhibitor H89, the Ang II effects on peak I(N)a and I-Ca were also absent In contrast, genetic knockout of CaMKII delta (CaMKII delta(-/-)) did not influence the Ang II-dependent increase in peak I-Ca and I-Na. On the other hand, Aug II enhanced I-Na inactivation, increased late I-Na and induced diastolic SR (sarcoplasmic reticulum) Ca leak (confocal Ca spark measurements) in a CaMKII delta-, but not PKA-dependent manner. Surprisingly, only the increase in diastolic SR Ca leak was absent in gp91phox(-/-) myocytes suggesting that Ang II regulates I-Na inactivation in a manner dependent on CaMKII- but not on NOX2. Finally, we show that Aug 11 increased the propensity for cellular arrhythmias, for which PKA and CaMKII contribute, both dependent on NOX2. Conclusion: Aug 11 activates PKA and CaMKII via NOX2, which results in disturbed Na and Ca currents (via PKA) and enhanced diastolic SR Ca leakage (via CaMKII). Oxidative activation of MA and CaMKII via NOX2 may represent important pro-arrhythmogenic pathways in the setting of increased Ang II stimulation, which may be relevant for the treatment of arrhythmias in cardiac disease. (C) 2014 Elsevier Ltd. All rights reserved.