CaMKII Mediates Digitalis - Induced Arrhythmias Running title :

Background Digitalis-induced Na accumulation results in an increase in Cai via the Na/Ca exchanger leading to enhanced SR Ca load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. Digitalis-induced increase in Cai could also activate 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 μmol/l ouabain increased contraction amplitude by 160 ± 5%. In the absence of electrical stimulation, ouabain promoted spontaneous contractile activity and Ca waves. Ouabain activated CaMKII (P-CaMKII) which phosphorylated its downstream targets, phospholamban (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 μmol/l KN93 but not by 2.5 μmol/l of the inactive analogue KN92. Similar results were obtained using the CaMKII inhibitor, AIP (1-2.5 μmol/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 content and Ca spark frequency, indicative of enhanced SR Ca leak. KN93 suppressed the ouabaininduced increase in Ca spark frequency without affecting SR Ca 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 ouabaininduced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca leak from the SR is the underlying mechanism involved.