Impacts of loss-of-function RyR2 mutations on Ca2+ signaling in non-cardiac and cardiac cells

[Speaker] Naoyuki Tetsuo:1
[Co-author] Nagomi Kurebayashi:1, Takashi Murayama:1, Takashi Sakurai:1
1:Department of Pharmacology, Juntendo University Faculty of Medicine, Japan

Background: Type 2 ryanodine receptor (RyR2) is the Ca2+ release channel on cardiac sarcoplasmic reticulum and plays a pivotal role in excitation-contraction coupling in the heart. Mutations in RyR2 have been linked to various types of ventricular arrhythmias including catecholaminergic ventricular tachycardia (CPVT), idiopathic ventricular fibrillation (IVF), long QT syndrome (LQTS), short coupled variant of Torsades de Pointes (scTdP). These mutations are supposed to develop arrhythmia via abnormal Ca2+ signaling in cardiac cells, but the cellular mechanisms have not been well understood. In this study, we aimed to characterize properties of IVF, LQTS and scTdP mutants by expressing RyR2 in non-cardiac (HEK293) and cardiac-derived HL-1 cells, to understand mechanism of various ventricular arrhythmias by RyR2 mutations.
Methods: Cytoplasmic and ER Ca2+ signals in HEK293 cells expressing exogenous homotetramer of wild type (WT) or mutant RyR2s were monitored with G-GECO1.1 and R-CEPIA1er, respectively. HL-1 cells, which constitutively express intrinsic WT RyR2, were infected with RyR2-IRES-mCherry baculovirus. Intracellular Ca2+ signals were monitored with Cal520 in cells in clusters showing rhythmic Ca2+ transients
Results and Discussion: When WT RyR2 was expressed in HEK293 cells, they showed periodic Ca2+ oscillations. Two of IVF-related RyR2 mutants exerted more frequent Ca2+ oscillations compared to WT (gain-of-function mutants). In contrast, two IVF-, two LQTS- and one scTdP-related mutants showed no Ca2+ oscillations (loss-of-function mutants). Expression of the gain-of-function mutants in HL-1 cells caused frequent Ca2+ waves during action potential induced Ca2+ transients. Among five loss-of-function mutants, three mutants exerted no Ca2+ waves with reduced amplitudes of the action potential-induced Ca2+ transients. Interestingly, the other two mutants exerted frequent Ca2+ wavelets in HL-1 cells. These results suggest that properties of heterotetramer of WT and mutant RyR2 channels may be different from that of homotetramer channel of mutant RyR2. Potential mechanisms of arrhythmia caused by loss-of-function type RyR2 mutation will be discussed.

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