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, Arest, was greater in CPVT mutants than in WT, and we found an inverse correlation between threshold Ca2+ER and Arest. In addition, lowering RYR2 expression increased the threshold Ca2+ER and a product of Arest, 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.