Enhanced CICR activity reduces ER Ca2+level in cells expressing CPVT-linked mutant RyR2

Type 2 ryanodine receptor (RyR2) is a cardiac Ca 2+ release channel in the endoplasmic reticulum (ER). Mutations in RyR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), which is considered to be associated with enhanced spontaneous Ca 2+ release. This spontaneous Ca 2+ release tends to occur when ER Ca 2+ ([Ca 2+ ] ER ) reaches a certain threshold level, and CPVT mutations are reported to lower this threshold. There are two explanations for this lowered threshold: the mutations increase sensitivity to luminal Ca 2+ or they enhance cytosolic [Ca 2+ ] ([Ca 2+ ] cyt )-induced Ca 2+ release (CICR) activity. However, no quantitative analysis of this issue has been performed so far. Here, we quantitatively explored how the change in CICR activity of RyR2 affects the threshold [Ca 2+ ] ER experimentally and by model-based simulation. Wild-type (WT) and CPVT-linked mutant RyR2s were expressed in HEK293 cells. [Ca 2+ ] cyt and [Ca 2+ ] ER measurements with Ca 2+ indicators revealed that CPVT RyR2 cells showed higher oscillation frequency and lower threshold [Ca 2+ ] ER in a mutation-specific manner compared with WT cells. The CICR activity of mutant RyR2s was assessed by Ca 2+ -dependent [ 3 H]ryanodine binding and parameter analysis. CICR activity at resting [Ca 2+ ] cyt , A 7.0 , was higher in CPVT mutants than in WT and a strong inverse correlation was found between threshold [Ca 2+ ] ER and A 7.0 . Interestingly, lowering RyR2 expression increased threshold [Ca 2+ ] ER , suggesting that the threshold [Ca 2+ ] ER depends on net Ca 2+ release rate via RyR2, a product of A 7.0 for each mutant and the density of RyR2 molecules. A model-based simulation successfully reproduced the [Ca 2+ ] cyt and [Ca 2+ ] ER changes. Interestingly, the CICR activity associated with specific mutations correlated well with the age of onset of the disease in CPVT patients carrying the mutations. Our data suggest that the reduction in threshold [Ca 2+ ] ER for spontaneous Ca 2+ release by CPVT mutation is explained by enhanced CICR activity without considering a change in the [Ca 2+ ] ER sensitivity of RyR2. Summary CPVT-linked RyR2 mutations are prone to induce spontaneous Ca 2+ release from ER, which is strongly associated with arrhythmias. Kurebayashi et al. quantitatively explore how the changes in CICR activity by RyR2 mutations affect spontaneous Ca 2+ experimentally and by model simulation.