Abnormal regulation of RyR2 by Ca2+/calmodulin-dependent protein kinase 2 (CaMKII) has been suggested as a cause of sarcoplasmic reticulum (SR) Ca2+ leakage and contractile dysfunction in heart failure. We hypothesized that CaMKII phosphorylation of RyR2 is crucial for heart failure development. We generated RyR2 knockin mice in which CaMKII phosphorylation site S2814 was mutated to alanine (S2814A) to prevent phosphorylation. Cardiac function and dimensions monitored by echocardiography were similar in S2814A and WT mice up to 12 months of age. WT (n=13) and S2814A mice (n=9) were subjected to transverse aortic constriction (TAC) to induce pressure overload. At 8 weeks after TAC, S2814A mice displayed a similar hypertrophic response and decrease in ejection fraction (EF) as WT mice. At 16 weeks after TAC, however, EF was significantly lower in WT (32.5 +/- 3.4%) compared to S2814A mice (43.0 +/- 2.9%) suggesting inhibition of heart failure development in the latter. This rescue effect was further verified by a lower lung-weight-to-tibia-length ratio and a decrease in expression levels of the cardiac stress genes ANF and BNP in S2814A mice compared to WT mice at 16 weeks after TAC. Ca2+ imaging in cardiomyocytes, isolated from S2814A and WT mice at 16 weeks after sham or TAC surgery, demonstrated an decreased incidence of spontaneous SR Ca2+ release (SCR) events in S2814A (29.7% of myocytes) compared to WT (61.3% of myocytes; P<0.01). Whereas CaMKII inhibitor KN93 reduced the incidence of SCR events in WT to 36.1% (P<0.01 vs. WT TAC), KN93 did not have an effect of SCR in S2814A myocytes (30.0%; P=NS vs. S2814A TAC). Together, our results demonstrate that blocking CaMKII phosphorylation of RyR2 prevents SR Ca2+ leak, and inhibits or delays the progression to congestive heart failure in S2814A mice.