Cytosolic Ca(2+)-dependent Ca(2+) release activity primarily determines the ER Ca(2+) level in cells expressing the CPVT-linked mutant RYR2

Type 2 ryanodine receptor (RYR2) is a cardiac Ca(2+) 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.