CONICET | Buscador de Institutos y Recursos Humanos

BackgroundDigitalis-induced Na accumulation results in an increase in Ca2 i via the Na/Ca2 exchanger, leading to enhanced sarcoplasmic reticulum (SR) Ca2 load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. A digitalis-induced increase in Ca2 i could also activate calcium-calmodulin kinase II (CaMKII), which has been shown to have proarrhythmic effects. Here, we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. Methods and ResultsIn paced rat ventricular myocytes (0.5 Hz), 50 mol/L ouabain increased contraction amplitude by 1605%. In the absence of electric stimulation, ouabain promoted spontaneous contractile activity and Ca2 waves. Ouabain activated CaMKII (p-CaMKII), which phosphorylated its downstream targets, phospholamban (PLN) (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 mol/L KN93 but not by 2.5 mol/L of the inactive analog, KN92. Similar results were obtained using the CaMKII inhibitor, autocamtide-2 related inhibitory peptide (AIP) (1 to 2.5 mol/L), and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca2 content and Ca2 spark frequency, indicative of enhanced SR Ca2 leak. KN93 suppressed the ouabain-induced increase in Ca2 spark frequency without affecting SR Ca2 content. Similar results were obtained with digoxin. In vivo, ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. ConclusionsThese results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca2 leak from the SR, is the underlying mechanism involved.