Motivation: Patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) have RyR2 mutations and are prone to delayed after depolarisations (DAD), triggered by Ca2+ waves during β- adrenergic stimulation. These arrhythmias are often seen during periods where the heart rate increases; therefore, our objective was to study the effects of various stimulation frequencies in the RyR2R4496C knock-in mouse model of CPVT. Methods: Ventricular cardiac myocytes were isolated from either control or RyR2R4496C knock-in mutant mouse hearts by enzymatic digestion and loaded with Fluo-5F. Cells were then voltage clamped at -50 mV and depolarised to 0 mV using a 50 ms pulse. Cells were paced at frequencies of 1, 3, and 5 Hz in the presence of 1 μM isoproterenol. SR Ca2+ content was measured using the application of 5 mM caffeine and 20 mM 2,3-butane-dione-monoxime, with integration of the Na+-Ca2+ exchange current. All animal experimentation was compliant with the 1986 Scientific Procedures Act (UK). Results: Our data show that SR Ca2+ content is reduced by the RyR2R4496C mutation at 1, 3 and 5 Hz (P<0.05). Furthermore, SR Ca2+ content also increases with rate in control cells (1 Hz vs. 3 and 5 Hz P<0.05). However, this relationship appears to be impaired in RyR2R4496C mutant cells, with only 1 Hz vs. 5 Hz showing an increase in SR Ca2+ content. Our work also shows that increasing the stimulation frequency reduces both the L-type Ca2+ current and systolic Ca2+ transient amplitude. In addition, the RyR2R4496C mutation does not alter other Ca2+ cycling parameters, such as, the rate constant of decay for the systolic Ca2+ transient. Conclusion: These experiments demonstrate the RyR2R4496C mutation lowers SR Ca2+ content at all frequencies of stimulation, and modifies the relationship between frequency of stimulation and SR Ca2+ content. These observations suggest that the mutated RyR2R4496C responds differently to increases in the pacing frequency. This abnormal response to high frequencies of stimulation could explain why spontaneous Ca2+ release occurs at high frequencies of stimulation. This work offers mechanistic insights into the function of RyR2 in health and disease, aiding the development of future therapeutics.