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

• Published in: The Journal of general physiology Vol. 154; no. 9; p. 1
• Main Authors: Kurebayashi, Nagomi, Murayama, Takashi, Ota, Ryosaku, Suzuki, Junji, Kanemaru, Kazunori, Kobayashi, Takuya, Ohno, Seiko, Horie, Minoru, Iino, Masamitsu, Yamashita, Fumiyoshi, Sakurai, Takashi
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 05.09.2022
• Series: Excitation–Contraction Coupling
• Subjects: Calcium (intracellular); Calcium - metabolism; Calcium release channels; Calcium signalling; Excitation-Contraction Coupling; HEK293 Cells; Humans; Intercellular signaling; Mutants; Mutation; Myocytes, Cardiac - metabolism; Oscillations; Ryanodine Receptor Calcium Release Channel - genetics; Ryanodine Receptor Calcium Release Channel - metabolism; Ryanodine receptors; Tachycardia; Tachycardia, Ventricular - genetics; Tachycardia, Ventricular - metabolism; Ventricle
• ISSN: 0022-1295; 1540-7748
• DOI: 10.1085/jgp.202112869

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.