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 Ca-2(+) release, which tends to occur when [Ca-2(+)](ER) reaches a threshold. Mutations lower the threshold [Ca-2(+)](ER) by increasing luminal Ca-2(+) sensitivity or enhancing cytosolic [Ca-2(+)] ([Ca-2(+)](cyt))-dependent activity. Here, to establish the mechanism relating the change in [Ca-2(+)](cyt)-dependent activity of RYR2 and the threshold [Ca-2(+)](ER), we carried out cell-based experiments and in silico simulations. We expressed WT and CPVT-linked mutant RYR2s in HEK293 cells and measured [Ca-2(+)](cyt) and [Ca-2(+)](ER) using fluorescent Ca-2(+) indicators. CPVT RYR2 cells showed higher oscillation frequency and lower threshold [Ca-2(+)](ER) than WT cells. The [Ca-2(+)](cyt)-dependent activity at resting [Ca-2(+)](cyt), A(rest), was greater in CPVT mutants than in WT, and we found an inverse correlation between threshold [Ca-2(+)](ER) and A(rest). In addition, lowering RYR2 expression increased the threshold [Ca-2(+)](ER) and a product of A(rest), and the relative expression level for each mutant correlated with threshold [Ca-2(+)](ER), suggesting that the threshold [Ca-2(+)](ER) depends on the net Ca-2(+) release rate via RYR2. Modeling reproduced Ca-2(+) oscillations with [Ca-2(+)](cyt) and [Ca-2(+)](ER) changes in WT and CPVT cells. Interestingly, the [Ca-2(+)](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 [Ca-2(+)](ER) for spontaneous Ca-2(+) release by CPVT mutation is explained by enhanced [Ca-2(+)](cyt)-dependent activity without requiring modulation of the [Ca-2(+)](ER) sensitivity of RYR2.