The brief opening mode of the mitochondrial permeability transition pore (mPTP) serves as a calcium (Ca²⁺) release valve to prevent mitochondrial Ca²⁺ (mCa²⁺) overload. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to mutations in the Ca²⁺ release channel complex of ryanodine receptor 2 (RyR2). We hypothesize that inhibiting the mPTP opening in CPVT exacerbates the disease phenotype. By crossbreeding a CPVT model of CASQ2 knockout (KO) with a mouse missing CypD, an activator of mPTP, a double KO model (DKO) was generated. Echocardiography, cardiac histology, and live-cell imaging were employed to assess the severity of cardiac pathology. Western blot and RNAseq were performed to evaluate the contribution of various signaling pathways. Although exacerbated arrhythmias were reported, the DKO model did not exhibit pathological remodeling. Myocyte Ca²⁺ handling was similar to that of the CASQ2 KO mouse at a low pacing frequency. However, increased ROS production, activation of the CaMKII pathway, and hyperphosphorylation of RyR2 were detected in DKO. Transcriptome analysis identified altered gene expression profiles associated with electrical instability in DKO. Our study provides evidence that genetic inhibition of mPTP exacerbates RyR2 dysfunction in CPVT by increasing activation of the CaMKII pathway and subsequent hyperphosphorylation of RyR2.