Background-Digitalis-induced Na ϩ accumulation results in an increase in Ca 2ϩ i via the Na ϩ /Ca 2ϩ exchanger, leading to enhanced sarcoplasmic reticulum (SR) Ca 2ϩ load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. A digitalis-induced increase in Ca 2ϩ 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 Results-In paced rat ventricular myocytes (0.5 Hz), 50 mol/L ouabain increased contraction amplitude by 160Ϯ5%. In the absence of electric stimulation, ouabain promoted spontaneous contractile activity and Ca 2ϩ 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 Ca 2ϩ content and Ca 2ϩ spark frequency, indicative of enhanced SR Ca 2ϩ leak. KN93 suppressed the ouabain-induced increase in Ca 2ϩ spark frequency without affecting SR Ca 2ϩ content. Similar results were obtained with digoxin. In vivo, ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. Conclusions-These 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 Ca 2ϩ leak from the SR, is the underlying mechanism involved. (Circ Arrhythm Electrophysiol. 2011;4:947-957.) Key Words: cardiotonic steroids Ⅲ arrhythmias Ⅲ CaMKII Ⅲ heart failure C ardiotonic glycosides selectively bind to and inhibit the sarcolemmal Na ϩ /K ϩ -ATPase and cause an increase in intracellular Na ϩ , which in the heart reduces Ca 2ϩ extrusion and/or increases Ca 2ϩ influx through the Na ϩ /Ca 2ϩ exchanger (NCX). This increase in Ca 2ϩ i leads to an increase in sarcoplasmic reticulum (SR) Ca 2ϩ load and to a positive inotropic effect, which explains, at least in part, their thera- Clinical Perspective on p 957peutic use for heart failure treatment 1 ; however, these compounds have associated arrhythmic/toxic effects that conspire against their extensive use in the clinical practice. 2 The arrhythmic effects have been proposed to occur when the SR Ca 2ϩ storage capacity is exceeded so that oscillations of release-uptake cycles arise to re-establish the Ca 2ϩ equilibrium between the cytosol and the SR. These transient increases in Ca 2ϩ i (Ca 2ϩ waves) activate a transient inward (depolarizing) current (I ti ), primarily mediated by the forward-mode ...

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