RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced Ca2+ release (SOICR)

Abstract

The cardiac ryanodine receptor (RyR2) governs the release of Ca2+ from the sarcoplasmic reticulum, which initiates muscle contraction. Mutations in RyR2 have been linked to ventricular tachycardia (VT) and sudden death, but the precise molecular mechanism is unclear. It is known that when the sarcoplasmic reticulum store Ca2+ content reaches a critical level, spontaneous Ca2+ release occurs, a process we refer to as store-overload-induced Ca2+ release (SOICR). In view of the well documented arrhythmogenic nature of SOICR, we characterized the effects of disease-causing RyR2 mutations on SOICR in human embryonic kidney (HEK)293 cells and found that, at elevated extracellular Ca2+ levels, HEK293 cells expressing RyR2 displayed SOICR in a manner virtually identical to that observed in cardiac cells. Using this cell model, we demonstrated that the RyR2 mutations linked to VT and sudden death, N4104K, R4496C, and N4895D, markedly increased the occurrence of SOICR. At the molecular level, we showed that these RyR2 mutations increased the sensitivity of single RyR2 channels to activation by luminal Ca2+ and enhanced the basal level of [3H]ryanodine binding. We conclude that disease-causing RyR2 mutations, by enhancing RyR2 luminal Ca2+ activation, reduce the threshold for SOICR, which in turn increases the propensity for triggered arrhythmia. Abnormal RyR2 luminal Ca2+ activation likely contributes to the enhanced SOICR commonly observed in various cardiac conditions, including heart failure, and may represent a unifying mechanism for Ca2+ overload-associated VT.

Fig. 1.

SOICR occurs in HEK293 cells expressing RyR2(wt) at elevated [Ca²⁺]_o. Stable inducible HEK293 RyR2(wt) cells were induced with tetracycline and loaded with 5 μM fura-2 acetoxymethyl ester in Krebs-Ringer-Hepes (KRH) buffer for 20 min at room temperature. Cells were perfused continuously with KRH buffer without (0 mM) or with 0.1, 0.2, 0.3, 0.5, or 1.0 mM CaCl₂ or 1.0 mM CaCl₂ plus 5 mM caffeine. (A) Single-cell fluorescent Ca²⁺ images in the presence (Upper) or absence (Lower) of 0.3 mM caffeine at various [Ca²⁺]_o (0-1.0 mM). (B) Fura-2 ratios of representative RyR2(wt) cells in the absence (green trace) and presence (blue trace) of 0.3 mM caffeine and a HEK293 parental cell expressing no RyR2 (pink trace). (C) The fraction (%, mean ± SEM) of cells that display Ca²⁺ oscillations in the presence (filled circle) and absence (open circle) of 0.3 mM caffeine. The total numbers of cells analyzed for Ca²⁺ oscillations were 563 (without 0.3 mM caffeine) and 237 (with 0.3 mM caffeine) from three to nine separate experiments. (D) Store Ca²⁺ content at various [Ca²⁺]_o, which was estimated by measuring the amplitude of caffeine-(5 mM) induced Ca²⁺ release from oscillating cells and normalized to the maximum level obtained at 1.0 mM [Ca²⁺]_o. Data shown are mean ± SEM from three to seven separate experiments.

Fig. 2.

Effects of CPVT RyR2 mutations on SOICR. Stable inducible HEK293 cells expressing RyR2 mutants were loaded with fura-2-acetoxymethyl ester and perfused continuously with various [Ca²⁺]_o, as described in the legend to Fig. 1. (A) Single-cell fluorescent Ca²⁺ images of R4496C cells at various [Ca²⁺]_o (0-1.0 mM). (B) Fura-2 ratios of a representative R4496C cell (blue trace) and RyR2(wt) cell (green trace) at various [Ca²⁺]_o.(C) Fraction (%, mean ± SEM) of N4104K (filled diamond), R4496C (filled circle), or N4895D (filled square) cells that display Ca²⁺ oscillations. The total numbers of cells analyzed for Ca²⁺ oscillations were 563 for RyR2(wt), 327 for N4104K, 378 for R4496C, and 191 for N4895D from three to nine separate experiments. (D) Frequency of Ca²⁺ oscillations and store Ca²⁺ level in HEK293 cells expressing RyR2(wt) in the absence (WT) and presence (Caff) of 0.3 mM caffeine and in HEK293 cells expressing the RyR2 mutants N4104K (NK), R4496C (RC), and N4895D (ND). Both the store Ca²⁺ content and the frequency of Ca²⁺ oscillations were determined at 1.0 mM [Ca²⁺]_o. Values were normalized to the WT level (100%). Data shown are mean ± SEM from three to seven separate experiments. RyR2(wt) and the mutant proteins were pulled down by GST-FKBP12.6 from the same amount of cell lysate and Western blotted with an anti-RyR antibody (E).

Fig. 3.

CPVT RyR2 mutations increase the sensitivity of single RyR2 channels to luminal Ca²⁺ activation. Single-channel activities of RyR2(wt) (A), N4104K (B), R4496C (C), and N4895D (D) were recorded in a symmetrical recording solution containing 250 mM KCl and 25 mM Hepes (pH 7.4). EGTA was added to either the cis or trans chamber to determine the orientation of the incorporated channel. The side of the channel to which an addition of EGTA inhibited the activity of the incorporated channel presumably corresponds to the cytoplasmic face. The Ca²⁺ concentration on both the cytoplasmic and the luminal face of the channel was adjusted to ≈45 nM. The luminal Ca²⁺ concentration was then increased to various levels by the addition of aliquots of CaCl₂ solution. The control current traces for RyR2(wt) or mutants are shown in a, whereas single-channel current traces at 300 μM luminal Ca²⁺ are depicted in b. The holding potential was -20 mV. Openings are downward. Po, arithmetic mean open time (To), and arithmetic mean closed time (Tc) are indicated on top. A short line to the right of each current trace indicates the baseline. (E) The relationships between Po and luminal Ca²⁺ concentrations of single RyR2(wt) (open triangle), N4104K (filled diamond), R4496C (filled circle), and N4895D (filled square) are shown. Data points shown are mean ± SEM from 21 RyR2(wt), 7 N4104K, 14 R4496C, and 10 N4895D single channels.

Fig. 4.

Effects of CPVT RyR2 mutations on [³H]ryanodine binding. [³H]ryanodine binding to cell lysate prepared from RyR2(wt) (open circle), N4104K (filled diamond), R4496C (filled circle), and N4895D (filled square) cells was carried out at ≈3nMCa²⁺, various concentrations of KCl (50-1,000 mM), and 5nM[³H]ryanodine (A), and at various concentrations of Ca²⁺ (≈0.2-0.1 mM), 100 mM KCl, and 5 nM [³H]ryanodine (B). [³H]Ryanodine binding shown in A was normalized to the binding measured in the presence of 800 mM KCl and 100 μM Ca²⁺, whereas [³H]ryanodine binding shown in B was normalized to the binding obtained at 100 mM KCl and 100 μMCa²⁺. Data points shown are mean ± SEM from three to seven experiments.

Fig. 5.

A proposed mechanism for CPVT associated with RyR2 mutations. The relationship between the threshold for SOICR and the SR-free Ca²⁺ level in normal (A) and CPVT SR (B) in the resting and stimulated states is schematically shown. The threshold for SOICR, which is primarily determined by RyR2, is depicted by a red bar. Note that the threshold for SOICR is reduced in the CPVT SR as a consequence of the RyR2 mutations. The SR free Ca²⁺ level, which is predominantly determined by CASQ2, is represented by the blue area. Note that the resting level of SR-free Ca²⁺ in the CPVT SR might have adapted to a reduced level due to the existence of SR autoregulation (33). An abrupt increase of SR-free Ca²⁺ as a result of stimulations by catecholamines or stresses is depicted by the yellow area. When the SR-free Ca²⁺ level reaches the SOICR threshold, SOICR occurs, leading to a large SR Ca²⁺ spillover, which in turn can generate DAD and triggered arrhythmia.