Novel Roles for Peroxynitrite in Angiotensin II and CaMKII Signaling

Chaoming Zhou,Dario A Vitturi,Derrick E Johnson,Swarna S Ramaswamy,Bruce A Freeman,Andy Hudmon,Edwin S Levitan,Franciso J Schopfer

doi:10.1038/srep23416

Abstract

Ca2+/calmodulin-dependent protein kinase II (CaMKII) oxidation controls excitability and viability. While hydrogen peroxide (H2O2) affects Ca2+-activated CaMKII in vitro, Angiotensin II (Ang II)-induced CaMKIIδ signaling in cardiomyocytes is Ca2+ independent and requires NADPH oxidase-derived superoxide, but not its dismutation product H2O2. To better define the biological regulation of CaMKII activation and signaling by Ang II, we evaluated the potential for peroxynitrite (ONOO−) to mediate CaMKII activation and downstream Kv4.3 channel mRNA destabilization by Ang II. In vitro experiments show that ONOO− oxidizes and modestly activates pure CaMKII in the absence of Ca2+/CaM. Remarkably, this apokinase stimulation persists after mutating known oxidation targets (M281, M282, C290), suggesting a novel mechanism for increasing baseline Ca2+-independent CaMKII activity. The role of ONOO− in cardiac and neuronal responses to Ang II was then tested by scavenging ONOO− and preventing its formation by inhibiting nitric oxide synthase. Both treatments blocked Ang II effects on Kv4.3, tyrosine nitration and CaMKIIδ oxidation and activation. Together, these data show that ONOO− participates in Ang II-CaMKII signaling. The requirement for ONOO− in transducing Ang II signaling identifies ONOO−, which has been viewed as a reactive damaging byproduct of superoxide and nitric oxide, as a mediator of GPCR-CaMKII signaling.

Highlights

Angiotensin II (Ang II) induces NADPH oxidase-dependent generation of partially reduced oxygen species to regulate CaMKIIδ activity and promote apoptosis, hypertrophy and downregulation of Kv4.3 K+ channels in cardiomyocytes[1,2,3,4,5,6]
The effect of ONOO− on CaMKIIδ methionine oxidation was determined and compared with a control consisting of neutral pH decomposition products and potential residual species used in the synthesis of ONOO− (nitrite (NO2−), nitrate (NO3−), H2O2)
A low signal for M281/282 oxidation was observed for no ONOO− treatment and treatment with low concentrations of ONOO− (0.2 and 2 μM), while 20 and 200 μM ONOO− enhanced methionine oxidation of CaMKIIδ (Fig. 1A), demonstrating concentration dependent redox modification of calmodulin-dependent protein kinase II (CaMKII) expose to ONOO−

Summary

Introduction

Angiotensin II (Ang II) induces NADPH oxidase-dependent generation of partially reduced oxygen species (superoxide, O2.−; hydrogen peroxide, H2O2) to regulate CaMKIIδ activity and promote apoptosis, hypertrophy and downregulation of Kv4.3 K+ channels in cardiomyocytes[1,2,3,4,5,6]. The latter effect is produced in neurons, possibly to contribute to hypothalamic function[7]. ONOO− participates in Ang II-induced CaMKII signaling by conducting experiments with purified CaMKII and cardiac and neuronal cells

Methods

Results

Conclusion