Abstract Body: The cardiac ryanodine receptor (RyR2) controls the release of Ca²⁺ from the sarcoplasmic reticulum (SR) and plays an essential role in excitation-contraction coupling in cardiomyocytes. Defective RyR2 function because of naturally occurring RyR2 variants can cause cardiac arrhythmias, cardiomyopathies, and sudden cardiac death (SCD). Gain-of-function (GOF) missense variants in the cardiac RyR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), which has been extensively characterized. Our team found that RyR2 loss-of-function (LOF) variants can cause a new entity of cardiac channelopathy, which is termed Ca²⁺ release deficiency syndrome (CRDS). CRDS is characterized by ventricular arrhythmias (VAs) and SCD, but a normal exercise stress testing (EST). Recently, a RyR2 C-terminal truncating variant, R4790Ter, was reported in a patient presenting with unexplained cardiac arrest. In vitro studies using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) showed that the RyR2-R4790Ter nonsense variant behaves like RyR2-GOF variants that are often associated with CPVT. However, it is unknown how a RyR2 C-terminal truncation that results in loss of channel function could lead to GOF phenotypes. Therefore, we have two aims for this project, 1) To identify and functionally characterize CRDS associated RyR2 LOF variants in HEK293 cells; 2) To understand mechanisms of CRDS associated with RyR2 nonsense truncating variants. The RyR2 truncating variants, including the R4790Ter, were generated by using the overlap extension method with PCR. We also generated a knock-in mouse model harboring the R4790Ter^+/- variant to investigate the susceptibility to VAs and potential arrhythmogenic mechanisms. We found that C-terminal RyR2 truncating variants are non-functional and can exert a dominant negative impact on the RyR2 WT. Mouse model carrying the RyR2-R4790Ter^+/- suppressed stress-induced VAs. On the other hand, R4790Ter^+/- increased the propensity for pacing-induced VAs using the CRDS-specific stimulation protocol consisting of long-burst, long pause, and short-coupled (LBLPS) ventricular extra-stimulus. RyR2-R4790Ter^+/- also promoted Ca²⁺ alternans in intact hearts compared with RyR2-WT, a hallmark of RyR2-CRDS variants. Therefore, our findings indicate that RyR2 C-terminal RyR2-R4790Ter^+/- truncating variant is associated with CRDS, but not CPVT.