The cardiac ryanodine receptor (RyR2), the principal Ca2+ release channel of the heart, is affected by patient mutations and catecholaminergic stress causing defective channel closure, irregular Ca2+ triggered action potentials (tAPs), and a ventricular arrhythmia syndrome (CPVT). Based on results obtained in heterologous overexpression models of CPVT mutant RyR2 channels, other groups suggested that catecholaminergic stimulation may lead to triggered activity by reducing the threshold for store overload-induced Ca2+ release. We have previously shown that heterozygous RyR2-R2474S (RyR2RS) knock-in mice reproduce the characteristic patient phonotype of CPVT. To clarify the physiological arrhythmia mechanism, we evaluated phospholamban deficient (PLN-/-;RyR2RS) cells with constitutive sarcoplasmic reticulum (SR) Ca2+ store overload. Double transgenic model: heterozygous RyR2RS knock-in mice were crossed with PLN-/- mice to produce RyR2RS;PLN-/- mice established in the C57Bl6 background. Electrophysiology: in vivo ECG as well as current-clamp combined with intracellular Ca2+ imaging of isolated ventricular myocytes (37C, fluo-3) was used to evaluate the influence of SR Ca2+ load on arrhythmogenic behavior. Comparing RyR2RS;PLN-/- vs. RyR2RS;PLN+/+ mice showed for PLN deficient RS cells: (1) significant QT interval shortening (60±2 ms vs. 68±2 ms, respectively; p<0.05); (2) significant action-potential shortening (APD90: 89±4 ms vs. 117±8 ms, respectively; p<0.05); (3) significantly accelerated Ca2+ transient decay (28±2 ms vs. 69±5 ms, p<0.001); and (4) significantly increased SR Ca2+ load (ΔF/F0: 9.7±1.6 vs. 5.3±1.4, respectively; p<0.001). (5) As expected, acute β-adrenergic stimulation with 30 nM isoproterenol resulted in no further increase of SR Ca2+ load in PLN-/- (ΔF/F0: RyR2RS;PLN-/- 8.1±2.7 vs. RyR2RS;PLN+/+ 7.0±1.7; n.s.). (6) Despite maximal, constitutive SR Ca2+ loading in RyR2RS;PLN-/- cardiomyocytes, the occurrence of tAPs during β-adrenergic stimulation was significantly decreased in RyR2RS;PLN-/- cells (RyR2RS;PLN+/+: 9 out of 12 cells (75%); RyR2RS;PLN-/-: 1 out of 10 cells (10%) had tAP; p<0.05). In summary, our data suggest that maximal upregulation of SERCA2a activity in PLN-/- (1) leads to increased SR Ca2+ load and accelerated cytosolic Ca2+ removal, which shortens the APD and the QT interval in vivo; and (2) decreases the propensity for tAPs in the context of a highly arrhythmogenic CPVT model with significantly increased diastolic SR Ca2+ leak. This suggests that, contrary to expectation, increased SR Ca2+ per se, at levels occurring during catecholaminergic stress, may not trigger APs under physiological conditions. An increased susceptibility of RyR2CaRS cells for tAPs might be blunted by accelerated SR Ca2+ uptake and altered diastolic Ca2+ handling dynamics.