Abstract Body: Gain-of-function mutations of the sarcoplasmic reticulum (SR) Ca²⁺ release channel, the ryanodine receptor (RyR2), are linked to the inherited arrhythmia syndrome catecholaminergic polymorphic ventricular tachycardia (CPVT). Increasing evidence suggests that RyR2 gain-of-function not only disturbs intracellular Ca²⁺ homeostasis but drives remodeling of cell signaling and ultrastructure that markedly contributes to the arrhythmogenic phenotype. It is well established that disturbed SR Ca²⁺ homeostasis can activate the endoplasmic reticulum (ER) stress response, yet whether RyR2 gain-of-function in CPVT drives ER stress is yet to be explored. The goal of our study was to determine the contribution of ER stress evoked by RyR2 gain-of-function to cardiac arrhythmogenesis. To test this, we created a new rat model of CPVT induced by RyR2-S2222L^(+/-) mutation. Simultaneous whole cell patch clamp and Ca²⁺ imaging demonstrated that under β-adrenergic stimulation, CPVT ventricular myocytes (VMs) exhibit a high propensity to spontaneous Ca²⁺ waves (SCWs) and delayed afterdepolarizations. Importantly, CPVT VMs showed increased XBP1 splicing as a marker of ER stress, as well as increased intra-SR redox stress measured using biosensor ER_roGFPiE. Assessment of ER stress proteins that contribute to SR redox status revealed upregulation of H₂0₂-producer enzyme ERO1α, with no compensatory change in expression of H₂O₂-degrader Peroxiredoxin-4 (PRDX4). Adenoviral overexpression of PRDX4 in CPVT VMs not only normalized SR redox status but attenuated Ca²⁺ mishandling, reducing RyR2 activity and the incidence of proarrhythmic spontaneous Ca²⁺ waves. Of note, immunofluorescence and biochemical studies suggest a direct interaction between RyR2 and PRDX4. To test whether targeting ER stress was protective at the whole heart level, we delivered AAV9-αMHC-PRDX4 to CPVT rats and performed ex vivo optical mapping. While CPVT hearts exhibited triggered activity and increased incidence of VT, sustained VT was prevented in PRDX4-injected hearts. Collectively, these data strongly suggest that ER stress contributes to the arrhythmogenic phenotype of CPVT. Targeting ER stress protein PRDX4 has promising therapeutic potential to normalize SR homeostasis, stabilize RyR2 activity and attenuate Ca²⁺-dependent arrhythmogenesis.