Background: Abnormal sarcoplasmic reticulum (SR) Ca ²⁺ release through ryanodine receptor 2 (RyR2) is a well-established mechanism of ventricular arrhythmias. Whether persistent RyR2 dysfunction can also initiate the structural remodeling characteristic of arrhythmogenic cardiomyopathy (ACM) remains unknown. We tested the hypothesis that chronic RyR2-mediated Ca ²⁺ leak is sufficient to couple electrical instability to progressive cardiomyopathy. Methods: We generated a rabbit carrying the severe gain-of-function RyR2-V2475F mutation and characterized disease progression using longitudinal cardiac phenotyping, continuous electrocardiographic monitoring, cellular electrophysiology and Ca ²⁺ imaging, histopathology, biochemical analyses, and single-nucleus RNA sequencing (snRNA-seq). Therapeutic potential was evaluated by continuous treatment with the selective RyR2 inhibitor ent -verticilide for two months. Results: RyR2-V2475F rabbits developed spontaneous ventricular tachyarrhythmias, sudden cardiac death, and progressive right ventricular (RV)-dominant fibrofatty cardiomyopathy recapitulating major features of human ACM. Malignant ventricular arrhythmias preceded overt structural remodeling and were associated at the cellular level with early spontaneous Ca ²⁺ release and early afterdepolarizations. Early disease was characterized by remodeling of excitation-contraction coupling with preserved SR Ca ²⁺ load and contractile function despite RyR2 dysfunction; upon disease progression, these compensatory changes were accompanied by SR Ca ²⁺ depletion, impaired RV contractility and reduced left ventricular filling. Single-nucleus transcriptomics identified early fibroblast expansion and predicted cardiomyocyte-centered paracrine signaling before advanced fibrofatty infiltration. Cardiomyocytes also exhibited an altered lipid-metabolic transcriptome and intracellular lipid accumulation, accompanied by enhanced mitochondrial Ca ²⁺ uptake. Selective RyR2 inhibition markedly reduced ventricular tachycardia burden and preserved RV contraction and LV filling during disease progression. Conclusions: Severe RyR2 gain-of-function can lead to both malignant ventricular arrhythmias and progressive arrhythmogenic cardiomyopathy in a large-animal model, establishing persistent intracellular Ca ²⁺ dysregulation as a mechanistic link between electrical and structural heart disease. The ability of selective RyR2 inhibition to suppress arrhythmias and preserve cardiac function identifies RyR2 as a potentially actionable therapeutic target in arrhythmogenic cardiomyopathy.

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