Identification of loss-of-function RyR2 mutations associated with idiopathic ventricular fibrillation and sudden death

Abstract

Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca2+ release deficiency syndrome (CRDS). Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca2+ release (SOICR) in human embryonic kidney 293 (HEK293) cells. E4146K also severely reduced cytosolic Ca2+ activation and abolished luminal Ca2+ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [3H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype (WT) channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.

Keywords: Disease mutations; Ryanodine receptor; Sarcoplasmic reticulum; Sudden cardiac death; Ventricular fibrillation; Ventricular tachyarrhythmias.

Figure 1. Identification and location of RyR2 mutations associated with IVF and SCD

(A) A schematic diagram of the linear sequence of RyR2. Major structural domains of RyR2 are depicted as solid blue boxes. The orange boxes indicate four disease-associated mutation clusters (mutation hotspots) in RyR2. IVF- and SCD-associated RyR2 mutations identified were listed underneath their corresponding domains. (B) Locations of RyR2 mutations in the three-dimensional structure of RyR2 (6JI0). The three-dimensional locations of RyR2 mutations, F2331S, G2337V, A3442E, I3476T, and R3570W have not been resolved and thus were not shown in the three-dimensional structure. The bindings sites for Ca²⁺, caffeine, and ATP are also shown.

Figure 2. Effects of RyR2 E4146K and G4935R mutations associated with sudden death on caffeine-induced Ca²⁺ release and their expression in HEK293 cells

HEK293 cells were transfected with RyR2 WT (A), E4146K (B), and G4935R (C). Fluorescence intensity of the fluo-3-loaded transfected cells was monitored continuously before and after each caffeine addition. The numbers (under the traces) indicate caffeine concentrations. Traces shown are from representative experiments. (D) Cumulative caffeine concentration–Ca²⁺ release relationships in HEK293 cells transfected with RyR2 WT, E4146K, and G4935R. Data shown are mean ± SEM (n=3). (E,F) HEK293 cells were transfected with RyR2 WT, E4146K, or G4935R. Cell lysates were prepared from these transfected cells and used for immunoblotting analysis. The same amount of cell lysate was used for immunoblotting using the anti-RyR2 antibody. Data shown are mean ± SEM (n=4, *P <0.05 vs. WT).

Figure 3. Effect of RyR2 mutations on caffeine-induced Ca²⁺ release in HEK293 cells

HEK293 cells were transfected with RyR2 WT or mutants. Fluorescence intensity of the fluo-3-loaded transfected cells was monitored continuously before and after each caffeine addition. The numbers (under the traces) indicate caffeine concentrations. Traces shown are from representative experiments (n=3). (A) RyR-WT. (B-Q) RyR2 mutants tested in the caffeine-induced Ca²⁺ release experiments.

Figure 4. Impact of RyR2 mutations on caffeine-induced Ca²⁺ release in HEK293 cells

HEK293 cells were transfected with RyR2 WT or mutants. Fluorescence intensity of the fluo-3-loaded transfected cells was monitored continuously before and after each caffeine addition. (A–C) Cumulative caffeine concentration–Ca²⁺ release relationships in HEK293 cells transfected with RyR2 WT or mutants. Note that the caffeine responses of these mutants are similar to that of RyR2 WT. Data shown are mean ± SEM (n=3).

Figure 5. The RyR2-E4146K mutaton abolishes store-overload induced Ca²⁺ release (SOICR) in HEK293 cells

Stable, inducible HEK293 cells expressing RyR2 WT or E4146K were loaded with 5 μM Fura-2 AM in KRH buffer. The cells were then perfused continuously with KRH buffer containing increasing levels of extracellular Ca²⁺ (0–2 mM) to induce SOICR. Fura-2 ratios of representative RyR2 WT (A) and E4146K (B) cells were recorded using single cell Ca²⁺ imaging. (C) The percentages of RyR2 WT (691 cells) and E4146K (466) cells that display Ca²⁺ oscillations at various extracellular Ca²⁺ concentrations. Note that no SOICR was detected in HEK293 cells expressing the E4146K mutant. (D) ER store Ca²⁺ content in RyR2 WT or E4146K mutant expressing HEK293 cells estimated by measuring the amplitude of caffeine (10 mM) induced Ca²⁺ release. Data shown are mean ± SEM (n=3–5, *P<0.05 vs. WT).

Figure 6. Effect of E4146K on luminal Ca²⁺ activation of single RyR2 channels

Single channel activities of the E4146K mutant (A) were recorded in a symmetrical recording solution containing 250 mM KCl and 25 mM HEPES (pH 7.4). The Ca²⁺ concentration on both the cytoplasmic and the luminal face of the channel was adjusted to ∼45 nM (panel a). The luminal Ca²⁺ concentration was then increased to various levels by an addition of aliquots of CaCl₂ solution (panels b–d). Recording potentials were −20 mV. Openings were downward and baselines indicated (short bars). Open probability (Po), mean open time (To), and mean closed time (Tc) are shown. (B) The relationships between Po and luminal Ca²⁺ concentrations (pCa) of single E4146K (open squares) mutant channels are shown. Data points shown are mean ± SEM from 7 E4146K single channels. The Po-luminal Ca²⁺ relationship (dashed line) of single RyR2 WT channels was taken from a previous study [46] where E4146K was part of the study.

Figure 7. Impact of the E4146K mutation on cytosolic Ca²⁺ activation of single RyR2 channels

Single channel activities of the E4146K mutant (A) were recorded in a symmetrical recording solution containing 250 mM KCl and 25 mM HEPES (pH 7.4) and in the presence of ∼45 nM luminal Ca²⁺ and various concentrations of cytosolic Ca²⁺ (panels a-c). (B) The relationship between Po and cytosolic Ca²⁺ concentrations (pCa) of single E4146K (open squares) mutant channels are shown. Data points shown are mean ± SEM from 14 E4146K single channels. The Po-cytosolic Ca²⁺ relationship (dashed line) of single RyR2 WT channels was taken from a previous study [46] where E4146K was part of the study.

Figure 8. The RyR2 LOF G4935R mutation has dominant negative effect on RyR2 WT

HEK293 cells were transfected with RyR2 WT (A) or co-transfected with RyR2 WT and G4935R (B). Fluorescence intensity of the fluo-3-loaded transfected cells was monitored continuously before and after each caffeine addition. (C) Cumulative caffeine concentration–Ca²⁺ release relationships in HEK293 cells transfected with RyR2 WT or co-transfected with RyR2 WT/G4935R. Data shown are mean ± SEM (n=3). (D) [³H]ryanodine binding to cell lysates prepared from HEK293 cells transfected with RyR2 WT or the G4935R mutant or co-transfected with RyR2 WT/G4935R was carried out at various Ca²⁺ concentrations (0.1 nM to 100 µM). Data shown are mean ± SEM (n=5 separate experiments for WT, 3 for RyR2 WT/G4935R, and 4 for G4935R. *P <0.05 vs.WT.).