Aims
Gain-of-function mutations in RYR2, encoding the cardiac ryanodine receptor channel (RyR2), cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Whereas, genotype–phenotype correlations of loss-of-function mutations remains unknown, due to a small number of analysed mutations. In this study, we aimed to investigate their genotype–phenotype correlations in patients with loss-of-function RYR2 mutations.
Methods and results
We performed targeted gene sequencing for 710 probands younger than 16-year-old with inherited primary arrhythmia syndromes (IPAS). RYR2 mutations were identified in 63 probands, and 3 probands displayed clinical features different from CPVT. A proband with p.E4146D developed ventricular fibrillation (VF) and QT prolongation whereas that with p.S4168P showed QT prolongation and bradycardia. Another proband with p.S4938F showed short-coupled variant of torsade de pointes (scTdP). To evaluate the functional alterations in these three mutant RyR2s and p.K4594Q previously reported in a long QT syndrome (LQTS), we measured Ca2+ signals in HEK293 cells and HL-1 cardiomyocytes as well as Ca2+-dependent [3H]ryanodine binding. All mutant RyR2s demonstrated a reduced Ca2+ release, an increased endoplasmic reticulum Ca2+, and a reduced [3H]ryanodine binding, indicating loss-of-functions. In HL-1 cells, the exogenous expression of S4168P and K4594Q reduced amplitude of Ca2+ transients without inducing Ca2+ waves, whereas that of E4146D and S4938F evoked frequent localized Ca2+ waves.
Conclusion
Loss-of-function RYR2 mutations may be implicated in various types of arrhythmias including LQTS, VF, and scTdP, depending on alteration of the channel activity. Search of RYR2 mutations in IPAS patients clinically different from CPVT will be a useful strategy to effectively discover loss-of-function RYR2 mutations.
Background: Type 2 ryanodine receptor (RyR2) is the Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ signals were monitored with Cal520 in cells in clusters showing rhythmic Ca 2+ transients Results and Discussion: When WT RyR2 was expressed in HEK293 cells, they showed periodic Ca 2+ oscillations. Two of IVF-related RyR2 mutants exerted more frequent Ca 2+ oscillations compared to WT (gain-of-function mutants). In contrast, two IVF-, two LQTS-and one scTdP-related mutants showed no Ca 2+ oscillations (loss-of-function mutants).Expression of the gain-of-function mutants in HL-1 cells caused frequent Ca 2+ waves during action potential induced Ca 2+ transients. Among five loss-of-function mutants, three mutants exerted no Ca 2+ waves with reduced amplitudes of the action potential-induced Ca 2+ transients. Interestingly, the other two mutants exerted frequent Ca 2+ 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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