Background-Approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias.Although abnormal Ca 2ϩ release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca 2ϩ remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca 2ϩ /calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. Methods and Results-Mice in which the S2814 Ca 2ϩ /calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca 2ϩ release events, resulting in reduced sarcoplasmic reticulum Ca 2ϩ load on confocal microscopy. These Ca 2ϩ release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca 2ϩ /calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery. Conclusions-Our results suggest that Ca 2ϩ /calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca 2ϩ release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure. (Circulation. 2010;122:2669-2679.)Key Words: arrhythmia Ⅲ calcium Ⅲ calcium-calmodulin-dependent protein kinase type 2 Ⅲ heart failure Ⅲ ryanodine receptor calcium release channel Ⅲ sarcoplasmic reticulum C ongestive heart failure (HF) is a leading cause of mortality and morbidity worldwide. Approximately 50% of HF patients die of sudden cardiac death (SCD) attributed to ventricular arrhythmias (Ͼ300 000 in the United States annually). 1,2 A large fraction of these arrhythmias are thought to be initiated by focal triggered mechanisms, such as spontaneous diastolic Ca 2ϩ release from cardiac myocyte ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR), which activates an arrhythmogenic depolarizing inward Na ϩ /Ca 2ϩ exchange (NCX) current. 3,4 Indeed, in HF there is enhanced diastolic SR Ca 2ϩ release and other changes in electrophysiological substrate that greatly enhance the propensity for triggered arrhythmias. Likewise, patients with inherited RyR2 point mutations exhibit catecholaminergic polymorphic ventricular tachycardia, a known cause of SCD with sensitivity to adrenergic conditions such as exercise or stress. 5,6 HF is a chronic hyperadrenergic state, and a prominent theory suggested that ␤-adrenergic activation of protein kinase A (PKA) destabilized ...

Rationale Increased activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) is thought to promote heart failure progression. However, the importance of CaMKII phosphorylation of ryanodine receptors (RyR2) in heart failure (HF) development and associated diastolic sarcoplasmic reticulum (SR) Ca2+ leak is unclear. Objective Determine the role of CaMKII phosphorylation of RyR2 in patients and mice with non-ischemic and ischemic forms of HF. Methods and Results Phosphorylation of the primary CaMKII site S2814 on RyR2 was increased in patients with non-ischemic but not with ischemic HF. Knock-in mice with an inactivated S2814 phosphorylation site were relatively protected from HF development following transverse aortic constriction (TAC) compared to wildtype (WT) littermates. After TAC, S2814A mice did not exhibit pulmonary congestion and had reduced levels of atrial natriuretic factor (ANF). Cardiomyocytes from S2814A mice exhibited significantly lower SR Ca2+ leak and improved SR Ca2+ loading compared to WT mice after TAC. Interestingly, these protective effects on cardiac contractility were not observed in S2814A mice following experimental myocardial infarction. Conclusions Our results suggest that increased CaMKII phosphorylation of RyR2 plays a role in the development of pathological SR Ca2+ leak and heart failure development in non-ischemic forms of HF such as transverse aortic constriction in mice.