Cytosolic Ca²⁺-dependent Ca²⁺ release activity primarily determines the ER Ca²⁺ level in cells expressing the CPVT-linked mutant RYR2

Type 2 ryanodine receptor (RYR2) is a cardiac Ca²⁺ release channel in the ER. Mutations in RYR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT is associated with enhanced spontaneous Ca²⁺ release, which tends to occur when [Ca²⁺]_ER reaches a threshold. Mutations lower the threshold [Ca²⁺]_ER by increasing luminal Ca²⁺ sensitivity or enhancing cytosolic [Ca²⁺] ([Ca²⁺]_cyt)-dependent activity. Here, to establish the mechanism relating the change in [Ca²⁺]_cyt-dependent activity of RYR2 and the threshold [Ca²⁺]_ER, we carried out cell-based experiments and in silico simulations. We expressed WT and CPVT-linked mutant RYR2s in HEK293 cells and measured [Ca²⁺]_cyt and [Ca²⁺]_ER using fluorescent Ca²⁺ indicators. CPVT RYR2 cells showed higher oscillation frequency and lower threshold [Ca²⁺]_ER than WT cells. The [Ca²⁺]_cyt-dependent activity at resting [Ca²⁺]_cyt, A_rest, was greater in CPVT mutants than in WT, and we found an inverse correlation between threshold [Ca²⁺]_ER and A_rest. In addition, lowering RYR2 expression increased the threshold [Ca²⁺]_ER and a product of A_rest, and the relative expression level for each mutant correlated with threshold [Ca²⁺]_ER, suggesting that the threshold [Ca²⁺]_ER depends on the net Ca²⁺ release rate via RYR2. Modeling reproduced Ca²⁺ oscillations with [Ca²⁺]_cyt and [Ca²⁺]_ER changes in WT and CPVT cells. Interestingly, the [Ca²⁺]_cyt-dependent activity of specific mutations correlated with the age of disease onset in patients carrying them. Our data suggest that the reduction in threshold [Ca²⁺]_ER for spontaneous Ca²⁺ release by CPVT mutation is explained by enhanced [Ca²⁺]_cyt-dependent activity without requiring modulation of the [Ca²⁺]_ER sensitivity of RYR2.