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.