Angiotensin II–Induced Oxidative Stress Resets the Ca 2 Dependence of Ca 2 –Calmodulin Protein Kinase II and Promotes a Death Pathway Conserved Across Different Species Julieta Palomeque, Omar Velez Rueda, Luciana Sapia, Carlos A. Valverde, Margarita Salas, Martin Vila Petroff, Alicia Mattiazzi Rationale: Angiotensin (Ang) II–induced apoptosis was reported to be mediated by different signaling molecules. Whether these molecules are either interconnected in a single pathway or constitute different and alternative cascades by which Ang II exerts its apoptotic action, is not known. Objective: To investigate in cultured myocytes from adult cat and rat, 2 species in which Ang II has opposite inotropic effects, the signaling cascade involved in Ang II–induced apoptosis. Methods and Results: Ang II (1 mol/L) reduced cat/rat myocytes viability by 40%, in part, because of apoptosis (TUNEL/caspase-3 activity). In both species, apoptosis was associated with reactive oxygen species (ROS) production, Ca 2 /calmodulin– dependent protein kinase (CaMK)II, and p38 mitogen-activated protein kinase (p38MAPK) activation and was prevented by the ROS scavenger MPG (2-mercaptopropionylglycine) or the NADPH oxidase inhibitor DPI (diphenyleneiodonium) by CaMKII inhibitors (KN-93 and AIP [autocamtide 2-related inhibitory peptide]) or in transgenic mice expressing a CaMKII inhibitory peptide and by the p38MAPK inhibitor, SB202190. Furthermore, p38MAPK overexpression exacerbated Ang II–induced cell mortality. Moreover, although KN-93 did not affect Ang II–induced ROS production, it prevented p38MAPK activation. Results further show that CaMKII can be activated by Ang II or H 2 O 2 , even in the presence of the Ca 2 chelator BAPTA-AM, in myocytes and in EGTA-Ca 2 –free solutions in the presence of the calmodulin inhibitor W-7 in in vitro experiments. Conclusions: (1) The Ang II–induced apoptotic cascade converges in both species, in a common pathway mediated by ROS-dependent CaMKII activation which results in p38MAPK activation and apoptosis. (2) In the presence of Ang II or ROS, CaMKII may be activated at subdiastolic Ca 2 concentrations, suggesting a new mechanism by which ROS reset the Ca 2 dependence of CaMKII to extremely low Ca 2 levels. (Circ Res. 2009;105:1204-1212.) Key Words: angiotensin II  CaMKII  apoptosis  reactive oxygen species E xperimental evidence indicates that a critical factor in the transition from compensated to noncompensated cardiac hypertrophy is myocyte cell loss by apoptosis. 1 The circulat- ing levels of Ang II are increased in heart failure and may constitute one of the major causes of cell death in this transition. 2 The apoptotic effects of Ang II have been reported to be mediated by different signaling molecules, eg, Ca 2+ , protein kinase (PK)C-, p38 mitogen-activated protein kinase (p38MAPK), or reactive oxygen species (ROS). 2–5 Recent reports have shown that activation of the multifunctional Ca 2+ /calmodulin– dependent protein kinase (CaMK)II is a common intermediate of diverse death stimuli–induced apo- ptosis in cardiac cells. 6 Supporting these results, we have demonstrated that CaMKII activation is a critical event in the signaling cascade that leads to apoptosis and necrosis occur- ring in reperfusion injury. 7 More importantly, we and others have shown that Ang II–induced apoptosis is caused by an increase in CaMKII activity mediated by ROS. 8,9 Recent exper- imental evidence further indicated that ROS-induced oxidation of methionine residues sustains CaMKII activity in the absence of Ca 2+ /calmodulin (Ca 2+ /CaM). However, this action requires previous binding of Ca 2+ /CaM to expose the autoinhibitory domain of CaMKII for oxidation. 8 Although these experiments suggest the concept that ROS can reset the Ca 2+ dependence of CaMKII activation, this possibility has never been tested. More- over, other findings have pointed to the role of phosphatase inhibition in ROS-induced CaMKII activation. 10 In spite of these important achievements in the understanding of the proapoptotic molecules activated by Ang II, the signaling cascade whereby the peptide produces apoptosis remains uncer- Original received July 1, 2009; revision received October 11, 2009; accepted October 13, 2009. From the Centro de Investigaciones Cardiovasculares, Facultad Medicina, Universidad Nacional de La Plata, Centro Científico Tecnolo ´ gico CONICET La Plata, Argentina. Correspondence to Dr Martin Vila Petroff, Centro de Investigaciones Cardiovasculares, Facultad de Medicina, La Plata, Argentina. E-mail mvila@aetos.med.unlp.edu.ar © 2009 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.109.204172 1204 Downloaded from http://ahajournals.org by on January 3, 2023