Ca2+-independent CaMKII activation by Angiotensin II: a novel cell death pathway

J. PALOMEQUE; JO. VELEZ RUEDA; L. SAPIA; CA. VALVERDE; MA. SALAS; MG. VILA PETROFF; A. MATTIAZZI

Nice, France

Congreso; Heart Failure Congress 2009 – ISHR European Section; 2009

European Society of Cardiology and International Society for Heart Research

CaMKII activation as well as reactive oxygen species (ROS) and angiotensin II (AngII) have been associated with cardiac apoptosis. Activity of the kinase as well as programmed cell death (PCD) are usually linked with abnormal Ca2+ homeostasis. Given that acute administration of AngII induces either no change or an increment in Ca2+i, in cardiac myocytes from rat and cat, respectively, our aim was to establish whether AngII produces PCD by activation of CaMKII in a Ca2+-dependent or –independent manner. For this purpose we used isolated myocytes from adult cats and rats cultured for 24 h with and without 1 µM AngII under unpaced conditions. We determined cell viability, apoptosis, CaMKII activity, Ca2+i and ROS production. AngII induced ≈40% of mortality in both species, at least in part by PCD (10.1±0.1% of TUNEL positive nuclei and 57.9±17% increase in caspase-3 activity), associated 1) with 42±9% increase in CaMKII activity, evaluated by the phosphorylation of the kinase (P-CaMKII) and of Thr17 residue of phospholamban (P-Thr17), a direct substrate of CaMKII and 2) with an increase in ROS production. KN-93, a selective CaMKII inhibitor, prevents CaMKII, and Thr17 phosphorylation, and the consequent AngII-induced PCD, but not the AngII-increased in ROS production. This would indicate that oxidative stress is upstream of the CaMKII activation. Unexpectedly, cells treated with AngII for 24 h from both species did not show any change in diastolic Ca2+, Ca2+ transient amplitude nor SR Ca2+ load. Furthermore no increase in Ca2+i was detected when diastolic Ca2+ was measured during the first 4 h of incubation with AngII, to exclude a possible transitory increase in Ca2+i at the beginning of the incubation period. Moreover, the IP3-receptor blocker, 2-APB, did not prevent AngII-induced cell death, indicating that IP3-induced Ca2+ release was not the source of Ca2+ for CaMKII activation. Finally, chelating Ca2+ with BAPTA-AM did not prevent AngII or H2O2–induced increase in P-CaMKII and cell death, and similar results were obtained under calmodulin inhibition with W-7. In vitro experiments also confirmed an increase in CaMKII activity produced by H2O2 in the absence of Ca2+ and calmodulin and in the presence of W-7. These results indicate that AngII-induced PCD cascade converges in both species in a common pathway mediated by ROS-dependent but previously unrecognized Ca2+-independent activation of CaMKII which results apoptosis