Arrhythmogenic mechanism of a novel ryanodine receptor mutation underlying sudden cardiac death

Abstract

Aims

The ryanodine receptor 2 (RyR2) is essential for cardiac muscle excitation–contraction coupling; dysfunctional RyR2 participates in the development of inherited arrhythmogenic cardiac disease. In this study, a novel RyR2 mutation A690E is identified from a patient with family inheritance of sudden cardiac death, and we aimed to investigate the pathogenic basis of the mutation.

Methods and results

We generated a mouse model that carried the A690E mutation. Mice were characterized by adrenergic-induced ventricular arrhythmias similar to clinical manifestation of the patient. Optical mapping studies revealed that isolated A690E hearts were prone to arrhythmogenesis and displayed frequency-dependence calcium transient alternans. Upon β-adrenoceptor challenge, the concordant alternans was shifted towards discordant alternans that favour triggering ectopic beats and Ca²⁺ re-entry; similar phenomenon was also found in the A690E cardiomyocytes. In addition, we found that A690E cardiomyocytes manifested abnormal Ca²⁺ release and electrophysiological disorders, including an increased sensitivity to cytosolic Ca²⁺, an elevated diastolic RyR2-mediated Ca²⁺ leak, and an imbalance between Ca²⁺ leak and reuptake. Structural analyses reveal that the mutation directly impacts RyR2–FK506 binding protein interaction.

Conclusion

In this study, we have identified a novel mutation in RyR2 that is associated with sudden cardiac death. By characterizing the function defects of mutant RyR2 in animal, whole heat, and cardiomyocytes, we demonstrated the pathogenic basis of the disease-causing mutation and provided a deeper mechanistic understanding of a life-threatening cardiac arrhythmia.

Graphical abstract

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Introduction

Ryanodine receptors (RyRs) are known as calcium-release proteins located on the sarcoplasmic reticulum (SR) membrane. RyRs mediate Ca²⁺-dependent activities, which allow for Ca²⁺ release from SR responsible for muscle contraction. So far, hundreds of variants have been found causing variety of diseases. Notably, the location of the mutation site generally has a great influence over protein function. RYR gene encodes a very large tetrameric structure protein (>2-MDa), which is also known as the largest ion channel. Wherefore, more researchers are devoted to understanding the pathophysiological mechanism of the disease, from the perspective of functional investigation on abnormal protein structure inducing gain/loss of function phenotype caused by variety mutations.

Catecholaminergic polymorphic ventricular tachycardia (CPVT), a type of stress-induced cardiac arrhythmia, mostly links to variants in the RyR2.¹ Catecholaminergic polymorphic ventricular tachycardia carried by the mutant RyR2 usually displays increased sensitivity to be activated which is so-called gain of function and generates delayed afterdepolarizations (DADs) that can trigger malignant Ventricular tachycardia (VT), identified as typical CPVT. The term ‘atypical CPVT’ is characterized by a combination of catecholamine-induced VT underlying QT interval prolongation, generated by prolonged action potential duration (APD) with frequent early afterdepolarizations (EADs) rather than DADs, which could be related to loss of function of RyR2.² Hence, mutations that alter the activation of RyR2 would be expected to result in cardiac dysfunction. Given its importance for normal RyR2 channel function, various regulatory molecules must co-operate with the tetrameric structure precisely. One of the well-characterized and controversial regulatory protein families is FK506 binding protein (FKBP) family. It is reported that FKBPs conduct controversial effects on RyRs,^3,4 using either RyR2 or FKBP12.6 knockout mice proving the association between RyR2 and FKBP12.6.^5,6 However, these mouse models are not carrying any RyR2 mutation, which is the most important causative factor in CPVT. In the present study, we identified a novel CPVT-causing mutation A690E with family inheritance located in “SPia kinase and RYanodine receptor 1” (SPRY1) domain (the FKBP binding domain in RyR2). We performed a comprehensive assessment of A690E mutation from clinical, genetic, functional, and structural perspectives.

Methods

Expanded methods are available in Supplementary material online, Supplemental Data.

Clinical studies

All clinical studies were approved by the Human Ethics Committee of Shanghai Tenth People’s Hospital, Tongji University School of Medicine (Approval Number: SHSY-IEC-KY-4.0/18-39/01). Written informed consent was obtained from each participant. The studies were performed in accordance with the Declaration of Helsinki.

Animal model

All animal studies were performed following guidelines from the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. All protocols were approved by the Institutional Animal Care and Use Committee of Shanghai Tenth People’s Hospital, Tongji University School of Medicine (Approval Number: SHDSYY-2018-3505). Knock-in mice were generated at the Shanghai Research Center for Model Organisms, an institute that is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). For differences between two experimental comparisons, two-tailed unpaired Student’s t-test was used. More than two experimental comparisons were assessed by one- or two-way analysis of variance (ANOVA) using Tukey’s honestly significant difference.

Results

Identification of a novel ryanodine receptor 2 mutation in a catecholaminergic polymorphic ventricular tachycardia patient

The proband (III-2 in Figure 1A) is a 52-year-old female who was diagnosed with a suspected case of CPVT at the Department of Cardiology, Shanghai Tenth People’s Hospital. (Detailed clinical description was shown in Supplementary material online, Supplemental Data.) The suspected CPVT symptoms include long-term sympathetic nerve excitement and frequent ventricular premature beats monitored by Holter. The 24 h ambulatory electrocardiogram (ECG) monitoring demonstrated her heart rates varying from 56 to 123 beats per minute and frequent premature ventricular contractions (PVCs) to 540 episodes per hour (see Supplementary material online, Table S3). Frequent PVCs and short episodes of polymorphic ventricular tachycardia (PVT) were observed (Figure 1B).

Next, a family pedigree investigation was carried out (Figure 1A). A mutation screening was performed using genomic DNA samples that were extracted from peripheral blood samples from the proband (III-2) and her sister (III-4). Unfortunately, her mother (II-2) and one nephew (IV-2) refused the genetic test, and another nephew (IV-3) was unable to be contacted. The mutation screening method was performed as described previously.⁷ A total of 10 mutations were identified within 9 genes. Next, Sanger sequencing was performed on the proband (III-2), her sister (III-4), and five arrhythmia-free, healthy control individuals (Figure 1C). Calsequestrin 2 (CASQ2) gene T66A was reported as innocent polymorphism also detected in the general population.⁸ Only C2069A in the RYR2 gene met the specification (Figure 1C), and this nucleotide substitution results in an amino acid at 690th position mutating from alanine (A) to glutamic acid (E) (A690E). Notably, this mutation was not reported in any database. The RYR2 gene is highly expressed in cardiomyocytes and has mutations that previously underlie CPVT.⁹ Thus, we hypothesized A690E in RyR2 is a novel CPVT-causing mutation.

Recurrence of catecholaminergic polymorphic ventricular tachycardia phenotypes in a knock-in mouse model carrier of the ryanodine receptor 2 A690E mutation

To date, more than 170 mutations in RyR2 have been linked to CPVT.¹ Of note, the A690E mutation we identified is not reported previously. To evaluate if the A690E mutation is associated with the pathologies of CPVT, we used CRISPR/Cas9 technology to generate knock-in mice. The strategy used, sequences of knock-in locus, and oligo donor DNA are described in Supplementary material online, Figure S1. Given that CPVT is an autosomal dominant disorder and the patient included in this study is heterozygous for the A690E mutation, we performed the investigation mainly in the heterozygous RyR2^A690E+/− mice. Generally, the RyR2^A690E+/− mice were indistinguishable from their WT littermates, manifested as similar body weights to WT mice from newborn to 10 months old (see Supplementary material online, Figure S2). No significant differences were observed in cardiac morphology between WT and RyR2^A690E+/− (see Supplementary material online, Figure S3). In general, no chronic consequences of the mutation on cardiac function from both genotypes were examined by echocardiography from 1 to 12 months of age (see Supplementary material online, Figure S4).

Interestingly, we found RyR2^A690E+/− mice were prone to death. We performed the Kaplan–Meier survival analyses during a 200 day period, the mortality rate of RyR2^A690E+/− mice was 20.2%, whereas the WT mice was only 1.1% (Figure 2A). To the recurrence of CPVT arrhythmia phenotypes in RyR2^A690E+/− mice model, we used a mouse-specific wireless telemetry device to record the ECG in live mice. We performed a classical pharmacological challenge testing for CPVT using caffeine (120 μg/g) and epinephrine (2 μg/g). Both WT and RyR2^A690E+/− mice displayed normal sinus rhythm prior to drug administration (Figure 2C). Two minutes after intraperitoneal injection of caffeine and epinephrine, PVCs and non-sustained VT were observed in RyR2^A690E+/− mice. Shortly after, the heart rate was dampened and soon turned into a mixture of PVT and bidirectional ventricular tachycardia (BVT). Around 1 h, heart rhymes rapidly deteriorated into ventricular fibrillation and asystole led to death (Figure 2C). Four out of 10 RyR2^A690E+/− mice died in the pharmacological challenge test; only transient sinus tachycardia was developed in WT mice (Figure 2B).

We further analysed the parameters of the recorded ECG. As shown in Figure 2D, WT and RyR2^A690E+/− mice exhibited no difference in QTc regardless of β-adrenergic stimulation, ruling out the possibility of long-QT syndrome, in which RYR2 is also one of the common causative genes. Mean values of PR interval were significantly shortened in RyR2^A690E+/− mice after stimulation (Figure 2E). Moreover, the cumulative incidence of non-sustained arrhythmias (arrhythmias duration ≤5 s, Figure 2F), sustained arrhythmias (arrhythmias duration >5 s, Figure 2G) and VT duration (the percentage of time in VT during entire >1 h recording period, Figure 2H) are all significantly increased in the RyR2^A690E+/− mice compared to WT, indicating that A690E mutation in RYR2 gene causes longer VT duration and more arrhythmias episodes, particularly the severe BVT and PVT, two classical CPVT arrhythmias.

In addition, we performed a treadmill test to evaluate whether exercise challenge could trigger arrhythmias in RyR2^A690E+/− mice. Both WT and RyR2^A690E+/− mice displayed normal sinus rhythm at resting condition prior to exercise (Figure 2I). In RyR2^A690E+/− mice, multiple PVCs were observed after running on treadmill, whereas only sinus tachycardia was observed in WT mice (Figure 2I), demonstrating a significant increase in PVCs compared to WT mice (Figure 2J). Since the running duration in RyR2^A690E+/− mice was short (see Supplementary material online, Figure S5), VT was not detected. Interestingly, in RyR2^A690E+/− mice, the heart rate was significantly higher compared to WT mice (Figure 2K), one of the ECG key features in CPVT response to exercise.¹⁰

In situ optical mapping of voltage and calcium in the RyR2^A690E+/− hearts response to β-adrenergic stimulation

We performed dual optical mapping with voltage- and Ca²⁺-sensitive dyes to analyse spatiotemporal V_m and [Ca²⁺]_i dynamics on Langendorff-perfused hearts. Firstly, the epicardial conduction patterns with 1μM isoproterenol (ISO) challenge and representative colour activation maps during apex pacing at 100 ms cycle length (CL) are presented in Figure 3A. In both genotypes, the anisotropic conduction patterns were similar, characterized by wavefront propagate from the apex (dark blue region) to the base (dark red region). We further quantified conduction velocities (CVs). As shown in Figure 3B, prior to ISO stimulation, both WT and RyR2^A690E+/− hearts have similar CVs. Conduction velocities were increased in the WT hearts with ISO stimulation as inotropic action, whereas in the RyR2^A690E+/− hearts, CVs were almost unchanged, a similar finding was previously reported in another mouse model of RyR2 mutation, P2328S, in which slow myocardial conduction and strong arrhythmic phenotype were present in hearts upon ISO challenge.¹¹

Next, action potentials (APs) and intracellular calcium transient (CaT) were optically mapped under sinus rhythm. Representative colour APD₈₀ maps and CaTD₈₀ maps were obtained from WT and RyR2^A690E+/− hearts at baseline and their response to ISO challenge. To demonstrate the spatial variability in AP and CaT, we also compared position traces at each spatial location (four points spanning the heart cross-section) in Figure 3C and F. The mean values (from the entire mapping field of hearts) for APD₈₀ and CaTD₈₀ and relative heterogeneity are detailed quantified. RyR2^A690E+/− hearts showed no significant difference in APD₈₀ values vs. WT response to ISO (Figure 3D), in agreement with no QTc change observed in vivo.¹² However, we observed more tissue heterogeneity of APD₈₀ in RyR2^A690E+/− hearts after ISO challenge (Figure 3E), especially at the site of left ventricular (spatial location #1 and trace #1 pointed by red arrowheads shown in Figure 3C). Isoproterenol shortened CaTD₈₀ values in both genotypes, but no difference in RyR2^A690E+/− hearts response to ISO vs. WT (Figure 3G), so as no difference in the CaTD₈₀ heterogeneity detected in both genotype (Figure 3H), illustrated Ca²⁺ handling properties are relatively conserved with genetically diverse backgrounds, at least under sinus rhythm.

We recorded the ECG simultaneously with V_m-[Ca²⁺]_i changes on isolated mouse hearts, to investigate the dynamics of epicardial propagation of arrhythmias and to demonstrate their focal origin and epicardial patterns. We found PVC and VT occurred more frequently upon ISO stimulation in RyR2^A690E+/− hearts (see Supplementary material online, Figure S6), consistent with our in vivo experiments. Meanwhile, we observed significant CaT alternans in RyR2^A690E+/− hearts (see Supplementary material online, Figure S7). CaT alternans was defined in a previous study: at least 10 consecutive beats showing the above 10% difference in amplitude of CaT between even and odd beats.¹³ The alternans phenomenon is less reported in homogeneous heart disease, but once has been reported in an induced pluripotent stem cell model¹³ and mice hearts of CPVT,^14,15 but lack of detailed mechanism elaboration. We recorded the V_m-[Ca²⁺]_i synchronously using an alternan-stimulation protocol (incrementally increasing pacing frequency CL 100–50 ms). Figure 4A–C displayed maps of CaT alternans magnitude and corresponding CaT traces at CL 50 ms with ISO challenge. Wild-type hearts display fewer CaT alternans at either CL (100 to 50 ms), characterized as homogeneous CaT spectral magnitude with consistent CaT traces in two locations (1 and 2, Figure 4A). Generally, most CaT alternans in RyR2^A690E+/− hearts are spatially concordant, the whole heart generated co-ordinated large/small amplitude CaT lasting in the consecutive beats, along with corresponding CaT traces from two locations (Figure 4B). Interestingly, we found certain CaT alternans display spatially discordant during pacing in RyR2^A690E+/− heart, showing two areas with opposite phases alternans, happening in the following consecutive heartbeats (Figure 4C). We quantified CaT alternans in alternans ratio, as shown in Figure 4D, RyR2^A690E+/− hearts with ISO challenge displayed a higher CaT alternans ratio between CL 70 to 50 ms compared to WT. Meanwhile, we also acquired the occurrence of CaT alternans in basal condition on both genotypes, Supplementary material online, Figure S8 showed a similar tendency. RyR2^A690E+/− hearts are more prone to generate CaT alternans than WT group. Moreover, our results also revealed that the addition of ISO has no inhibition effect on CaT alternans occurrence. And only with ISO stimulation in RyR2^A690E+/− hearts represented obvious regional heterogeneity, manifested as spatially discordant CaT alternans, which confirms the finding in another study.¹⁶

A previous study had demonstrated that mutation-caused functional alterations on RyR2 were the pivotal candidate, and RyR2 Ca²⁺ release refractoriness was the key causative factor for alternans.¹⁷ To test the effect of mutation on RyR2 Ca²⁺ release refractoriness in isolated RyR2^A690E+/− hearts, an S1S2 protocol was used (Figure 4E and F). This protocol consisted of trains of CL 100 ms pacing (S1) followed by a premature stimulus (S2) with stepwise shortening (2 ms) of the S1S2 interval. RyR2^A690E+/− hearts showed reduced recovery of CaTs (A2/A1 ratio) at S1S2 intervals of CL 54–64 ms (Figure 4G). As the S1S2 interval was reduced in steps, the A2/A1 ratio diminished until no measurable Ca²⁺ was released in response to the S2 stimulus. We used this interval as a measure of the RyR2 Ca²⁺ release refractory period. The Ca²⁺ release refractory period was increased in RyR2^A690E+/− hearts, compared with WT (Figure 4H). The above optical mapping experiments show that prolongation of Ca²⁺ release refractoriness, especially at high pacing frequency in RyR2^A690E+/−, which may explain the increased occurrence of alternans in this situation.

Interestingly, we did not observe APD alternans underlying CaT alternans occurrence in both genotypes (see Supplementary material online, Figure S9), which is reasonable that CaT alternans could develop irrespective of AP dynamics,¹⁸ and Ca²⁺ alternans is the driving reason in cardiac alternans. Thus, we quantified the change of APD₈₀ and relative APD heterogeneity during stimulation protocol. Supplementary material online, Figure S9B showed no significant APD₈₀ change among groups upon frequency increase. Interestingly, the APD heterogeneity of RyR2^A690E+/− hearts before ISO application were increasing vs. WT at high pacing frequency (CL 70–50 ms), and RyR2^A690E+/− hearts after ISO challenge maintained greater APD heterogeneity during CL 90–50 ms pacing vs. WT + ISO group (see Supplementary material online, Figure S9C). The formation of APD heterogeneity, which can form a vulnerable substrate for wave break.¹⁹

We also discovered that CaT alternans appear more severe during VT in the RyR2^A690E+/− heart, behaved as a significant amplitude difference between the odd and the even CaT (Figure 5A and B), and regions of heart are weakly coupled and evolve towards opposite alternans phase. The presence of spatially discordant alternans has been believed to be causally associated with ventricular arrhythmia.²⁰ Therefore, we investigated the relationship among locations of spatially discordant alternans and re-entrant activity in RyR2^A690E+/− heart. Figure 5A shows regional CaT trace (locations 1–3), revealing the transition from normality to spatially discordant alternans corresponding to the evolution of pacing-induced VT in the RyR2^A690E+/− heart with ISO challenge. Figure 5B and C shows zoomed views of traces and phase maps of several beats, in which beat (i) characterized as normal conduction direction by pacing at apex; beat (ii) appears as alternans induced the first ectopy; beat (iii) evolved a spatially discordant alternans-induced macro-reentry, which characterized as counterclockwise rotor activity. We found ectopy or re-entry activity starts from location #2 and has the most serious alternans (Figure 5C), which reflected the arrhythmogenic effect of alternans.

Ca²⁺ and electrophysiological homeostasis disorders in RyR2^A690E+/− cardiomyocytes

Next, we analysed adrenaline-induced Ca²⁺ handling and electrophysiological abnormalities in isolated RyR2^A690E+/− cardiomyocytes. First, we assessed the arrhythmogenicity of Ca²⁺ signals in freshly isolated myocytes following 1 Hz field-stimulated with ISO challenge by confocal microscopy. As shown in Figure 6A and B, upon ISO challenge, RyR2^A690E+/− cardiomyocytes have heightened propensity to generate frequent abnormal CaTs, manifested as diastolic premature Ca²⁺ transients (PCT, abnormal CaT occurred during diastolic phase) and Ca²⁺ oscillations (more than three consecutive peaks where the amplitude does not decay to the basal). We also examined the occurrence of the SR spontaneous Ca²⁺ release events following cessation of pacing, as shown in Supplementary material online, Figure S10, attenuated multiple spontaneous Ca²⁺ release, including spontaneous Ca²⁺ waves, afterdepolarizations induced trigger activities (TAs), and Ca²⁺ oscillations, were observed in RyR2^{A690E +/−} myocytes with ISO stimulation. In addition, we examined RyR2^A690E+/− myocytes whether β-adrenergic stimulation leading to pro-arrhythmic afterdepolarizations. Representative APs traces are shown in Figure 6C; hybridized small DADs, EADs, and TA have more occurred in RyR2^A690E+/− myocytes. Cumulative incidences of the above pro-arrhythmic disorders were summarized in Figure 6D–F, indicating RyR2^A690E+/− myocytes were prone to generate electrophysiological disorders, mainly in the form of DADs. These hybridized electrophysiological abnormalities will increase the spatial and temporal dispersion of AP, leading to cardiac arrhythmia.

A690E mutation contributes instability of ryanodine receptor 2 channel

To further examine the effect of A690E mutation on the channel gating, we quantified the rate of spontaneous RyR2 openings by measuring spontaneous Ca²⁺ sparks. Representative confocal line scan Ca²⁺ sparks images and corresponding three-dimensional surface plots are shown in Figure 7A and B. We summarized spark frequency in Figure 7C, compared to WT, RyR2^A690E+/− myocytes exhibited a significant increase in spark frequency, indicating unstable RyR2 Ca²⁺ release activity. Figure 7D–F showed Ca²⁺ spark characteristics, as amplitude (peak F/F0), full width at half maximum, and full duration at half maximum were indistinguishable in cells of either genotype with or without ISO.

Hyperactive RyR2 channel should be responsible for abnormal spontaneous Ca²⁺ release events. [³H]ryanodine binding has been widely used as a functional assay for RyR2 channel activities. We performed [³H]ryanodine binding to whole heart homogenates from WT and RyR2^A690E+/− mice. Supplementary material online, Figure S11A shows that RyR2^A690E+/− had a marked effect on the channel sensitivity to cytosolic Ca²⁺ activation, and relatively EC₅₀ values were significantly decreased in RyR2^A690E+/− compared to WT (see Supplementary material online, Figure S11B). Collectively, consistent with preceding Ca²⁺ dynamic studies, results manifested the A690E mutation induces an increased RyR2 activity.

To quantify the concrete SR Ca²⁺ leak, we used a general protocol shown in Figure 7G.²¹ Cardiomyocytes were firstly stimulated for at least tens of seconds at 1 Hz with normal Tyrode solution to maintain the cytoplasmic Ca²⁺ to a steady state and then paused pacing rapidly switched perfusate to 0 Na⁺, 0 Ca²⁺ Tyrode solution containing 1 mmol/L tetracaine; lastly, 10 mmol/L caffeine was added to quantify SR Ca²⁺ load, which is the difference fluorescence between the basal and peak [Ca²⁺]_i (Figure 7H). Quantification of SR Ca²⁺ leak is the tetracaine-induced diastolic Ca²⁺-shift (Figure 7I). We found that manifest decrease of SR Ca²⁺ load because of increased SR Ca²⁺ leak in RyR2^A690E+/− myocytes compared to WT. It reveals that the leakage caused by the A690E mutation favours uncontrollable Ca²⁺ release. We also quantified the integral of caffeine induced CaT (area under the curve);³ there was no significant difference between RyR2^A690E+/− and WT myocytes (see Supplementary material online, Figure S12A). We further analysed the fractional release (calculated as the ratio between the amplitudes of electrically evoked CaT and caffeine-evoked CaT); RyR2^A690E+/− myocytes showed slightly increased fractional release, compared to WT (see Supplementary material online, Figure S12B), indicated that increases Ca²⁺ release at a given SR Ca²⁺ load. Summary data reveal that the A690E mutation had no effect on the I_Ca density–voltage relationship relative to WT myocytes (see Supplementary material online, Figure S13). Similar results were also reported in mutations R420Q,²² F2483I, and Q4201R.²³ Recently, Fernández-Morales et al.³ have demonstrated that suppressed and down regulated L-type calcium channels could act as a compensation for FKBP-RyR destabilization, which results in lower SR Ca²⁺ content and Ca²⁺ leak in human stem cells-derived N771D mutant cardiomyocytes.

Recently, high-resolution structures of human RyR2 were determined by cryo-electron microscopy.²⁴ The A690E mutation is located in one of the RyR2 structural domains named ‘SPRY1’ (blue ribbon), a domain that directly interacts with FKBP (orange ribbon); detailed analyses reveal the A690E is located directly in the binding interface (Figure 7J and K). To further analyse the effect of the mutation on the protein–protein interaction, we truncated the RyR2 structure (PDB ID: 7U9R) to emphasize sequence around FKBP binding site, beside the SPRY1 domain 541–809, other domains including 1248–1316 (SPRY3), 1414–1848 (SPRY3 and handle domain), and 2105–2182 (helical domain), highlighted in gold ribbon in Figure 7J and K. The truncated structure was chosen to ensure that the binding interface between SPRY1 and FKBP where the A690E mutation locates was included, and the entire structure was large enough to mimic the whole RyR2 environment. We employed two different computational approaches, DUET²⁵ and MAESTRO,²⁶ to predicate the effects of missense mutations on protein stability. The DUET reported the predicted free energy changes (ΔΔG) of −1.145 kcal/mol, −0.29 kcal/mol, and −0.847 kcal/mol for mCSM, SDM, and DUET methods, respectively, which indicates that the structure was destabilized by the mutation of A690E. In comparison, the MAESTRO reported the ΔΔG _pred. value of 0.149 kcal/mol and C_pred. 0.877, which indicates the destabilizing effect of the mutation, is consistent with the DUTE results. In conclusion, two different prediction approaches showed that the structure becomes more unstable due to the A690E mutation. Next, we examined whether the binding of FKBP to RyR2 is generally altered as a result of A690E mutation, we implemented a GST pull down assay. Blots of FKBP associated with RyR2 in SR vesicles suggest that GST–FKBP displayed a strong interaction with RyR2 in WT, but the interaction significantly reduced in RyR2^A690E+/− (Figure 7L and 7M), which is consistent with our deduction of our structure analysis. We further used a drug S107 (a specific stabilizer of RyR2/FKBP12.6 complex),²⁷ to test pharmacologic prevention of A690E-RyR2 instabilizing in live mice using the osmotic pumps delivery method, and the therapeutic effect of the rescue experiment is relatively obvious (see Supplementary material online, Figure S14). We also examined the expression level of FKBP and other SR calcium release unit proteins by western blot; our data revealed that the A690E mutation had no effect on the relative protein expression (see Supplementary material online, Figure S15A and B). And, the A690E mutation did not alter the protein levels of two protein kinases, PKA and CaMKII (see Supplementary material online, Figure S15C–F). In addition, phosphorylation levels of RyR2 by three site- and phosphorylation-specific antibodies (anti-Ser-2814P, anti-Ser-2808P, and anti Ser-2030P) were detected, and western blot analysis showed that the A690E mutation displayed similar RyR2 phosphorylation changes, compared with WT, under resting condition even with ISO challenge (see Supplementary material online, Figure S15G–J). Previous studies have observed similar results, both RyR2 mutations P2328S and G230C did not interfere RyR2 phosphorylation levels.^28,29 Moreover, no difference in RyR2 phosphorylation between FKBP^−/− and FKBP^+/+ mice.³⁰

Predisposing cellular calcium transient alternans in RyR2^A690E+/− cardiomyocytes

We have observed CaT alternans in whole hearts; next, we assessed the response of electrical stimulation-evoked cellular CaT alternans and their corresponding effects on cardiomyocyte contractility. Beat-to-beat CaT alternans appeared when stimulation frequency increased, so as contraction amplitude alternans. Figure 8A shows cellular CaT recordings at 4 and 4.5 Hz, in which frequency CaT alternans emerge in RyR2^A690E+/− myocytes. It also reveals CaT alternans ratio increase as frequency accelerates in RyR2^A690E+/− myocytes. Figure 8B shows increasing the proportion of RyR2^A690E+/− cardiomyocytes with CaT alternans accompanied a decreased threshold frequency for Ca²⁺ alternans (Figure 8C). Similar results are observed in WT and RyR2^A690E+/− myocytes without ISO simulation (see Supplementary material online, Figure S16).

CPVT is mainly induced by stress or exercise, in which β-adrenergic stimulations elevate heart rate. Evidence suggests that a major component of alternans is due to a disruption in the cycling of Ca²⁺ at rapid rates that when heart rate is faster than the ability of a cell to release and reuptake Ca²⁺, alternans will occur.¹⁷ Accordingly, we compared properties of CaT at different pacing rates (1 Hz, basal frequency, and 4 Hz, in which CaT alternans emerge in RyR2^A690E+/− myocytes). Under 1 Hz pacing without ISO simulation, RyR2^A690E+/− myocytes behaved similarly to WT myocytes in parameters ‘Time-to-Peak’ (see Supplementary material online, Figure S17A) and ‘Decay-Time’ (see Supplementary material online, Figure S17B). We next analysed CaT at 4 Hz pacing, there was no difference in the value of time-to-peak (see Supplementary material online, Figure S17C). Interestingly, as stimulation rate increased, slower decay-time of the CaT (see Supplementary material online, Figure S17D) was evoked in RyR2^A690E+/− myocytes, and such frequency-dependent CaT duration change was indicated once in classical mutant RyR2^R4496C cardiomyocytes, proved to be a pro-arrhythmia behaviour.³¹ Decay-time as known reflecting SR Ca²⁺ reuptake rate, has been previously indicated that decreasing reuptake rate could be linked with promotion of CaT alternans.³² Meanwhile, the diastolic Ca²⁺ fluorescence measured in the same cells raised progressively with increasing pacing rate (defined as Δdiastolic Ca²⁺, the difference between diastolic Ca²⁺ at 4 and 1 Hz), and our results revealed an increased Δdiastolic [Ca²⁺]_i level in RyR2^A690E+/− myocytes, indicating increased SR Ca²⁺ leak as pacing raised (see Supplementary material online, Figure S17E). Under the same stimulus condition with ISO induction, RyR2^A690E+/− myocytes showed similar CaT trace to WT (Figure 8D) at 1 Hz pacing, behaved as no difference in time-to-peak (Figure 8E) and decay-time (Figure 8F). However, at 4 Hz during ISO stimulation, the difference in properties of CaT were more pronounced in RyR2^A690E+/− myocytes (Figure 8G). This CaT duration prolongation was associated with both slower time-to-peak (Figure 8H) and reduced decay-time (Figure 8I). Importantly, during ISO stimulation, Δdiastolic Ca²⁺ level was remarkably elevated in RyR2^A690E+/− (Figure 8J). A previous study had suggested that increased [Ca²⁺]_i resulted in the occurrence of CaT alternans, as the precursor of pro-arrhythmogenic calcium dysregulation at the cellular level.³³ Thus, both diastolic Ca²⁺ or CaT duration were altered in such a manner that it could explain the results of decreased threshold frequency for alternans in RyR2^A690E+/− myocytes.

Discussion

Previously studies have reported that mutations caused hyperactive RyR2 usually disturbing Ca²⁺ homeostasis (hypo-actively facilitating Ca²⁺ release from SR into cytosol). However, given the diversity of clinical phenotype and RyR2 channel functions, it is unclear in terms of defining an exact molecular basis for the leak and how alterations in RyR2 channel activity lead to cellular and ultimately to heart dysfunction. Disease-linked mouse models have address some questions. R4496C is the first RyR2 transgenic mouse model that recapitulate the main aspects of human CPVT;³¹ knock-in R4496C mice displayed increased RyR2 activities and channel sensitivity to Ca²⁺. Mouse hearts carry P2328S heterpzyotic mutation showed non-sustained and sustained VTs with regular stimuli and programmed electrical stimulation. Hearts carry P2328S homozygotic mutation showed higher incidences of non-sustained and sustained arrhythmogenesis. P2328S homozygous myocytes behaved more severe alterations in Ca²⁺ handling, corresponding graver arrhythmias phenotype in hearts isolated from the homozygous mice than heterpzyotic mutation, indicating a gene dosage effect of the P2328S mutation.³⁴ Mice with R2474S heterozygous mutation exhibited spontaneous generalized tonic-clonic seizures, exercise-induced ventricular arrhythmias, and sudden cardiac death;³⁵ R2474S sensitized the RyR2 channel to activation by luminal Ca²⁺ and in turn induces spontaneous Ca²⁺ release.³⁶ Mutation R176Q caused cardiac dysfunction and catecholamine-dependent arrhythmias and support an overlap between arrhythmogenic right ventricular dysplasia and CPVT.³⁷ Recently, VTs under stress conditions were observed in R420Q knock-in mice; this mutation also impaired the binding between RyR2 and junctophilin-2, defected nanoscale disarrangement of the dyad,²² and the mutation combines with adrenergic stimulation caused pathological conditions imbalanced Ca²⁺ release and uptake in R420Q, which are critical for arrhythmias occurrence.³⁸

Up to date, researchers have identified four mutations in the SPRY1 domain (see Supplementary material online, Figure S18A): D708N in RyR1 (equivalent D720 in RyR2),³⁹ N759D in RyR1 (equivalent N771 in RyR2),⁴⁰ R739H in RyR2,⁴¹ and recently, I784F mutation in RyR2.⁴² We pinpoint the four mutation sites in the structure, as shown in Supplementary material online, Figure S18B; only R739H is close to the FKBP binding site but has a farther distance to FKBP compared to A690E; analyses by DUET and MAESTRO confirmed that R739H has little effect on the ΔΔG. There are several controversial opinions about the RyR-FKBP binding. Priori and Chen⁴³ claimed FKBP binding is critical for selected mutations rather than applied to any mutation. To our best knowledge, there is no disease-causing mutation at place where directly impacts RyR2–FKBP molecular interaction. Here, we reported a novel CPVT-linked RyR2 mutation that identified from patient; the A690E mutation is located in the SPRY1 domain of RyR2 that immediately interacts with FKBP. This integrated study combining human, animal, organ, and cardiomyocyte data on the RyR2-A690E CPVT mutation is the first comprehensive description of mutation interfered with the RyR2–FKBP interaction caused RyR2 dysfunction, which was responsible for Ca²⁺ handling defect and occurrence of arrhythmia.

Organ-level synchronization of V_m and [Ca²⁺]_i signal recording integrated cardiac electrophysiology and Ca²⁺ handling response to β-adrenergic stimulation and further elaborated the arrhythmias mechanism. We captured isolated RyR2^A690E+/− hearts preferable to occur high pacing-rate induced sustained VT, underlying faster heart rhythm and serious CaT amplitude alternans. We refer to the CaT alternans generation protocol; as expected, CaT alternans mostly appear in all RyR2^A690E+/− hearts. Our results proved that alternans can be promoted by mutations that cause RyR2 to become leaky. We found similar alternans occurrence in RyR2^A690E+/− hearts no matter whether applied with ISO, as research indicated that once alternans is established and stabilized spatially in the well-coupled heart, ISO is not as effective in terminating the alternans,¹⁶ indicate that functional alterations on RyR2 and relative functional effects on RyR2 refractoriness are the pivotal candidate.⁴⁴ It is so-called ‘3R theory’, indicating that Ca²⁺ alternans emerge determined by three critical properties of the calcium release unit network: randomness, refractoriness, and recruitment.⁴⁴ Ca²⁺ alternans occur as the heart rate increases. The most direct effect of a fast heart rate is making RyR2 in the refractory state after firing during the previous beat. In normal mice, alternans happened only depending on the interval between two consecutive twitches, while SERCA could providentially replenish the SR with Ca²⁺. Enhancement of alternans phenomenon in RyR2-A690E indicates that more spontaneous Ca²⁺ release during diastole, an imbalance between Ca²⁺ leak and Ca²⁺ reuptake results in SR Ca²⁺ depletion, and a possible alteration in RyR2 refractoriness could account for this phenomenon. However, under basal conditions, the properties of CaT were generally similar, which further supports the lack of contractile impairment in CPVT patients. Our study shows that RyR2^A690E+/− displayed arrhythmogenic activity related to Ca²⁺ alternans while they are fast frequency electrically stimulated, thus mimicking human CPVT (conditions with adrenergic stimulation, which, among other effects, increases heart rate) and demonstrating that A690E was at the origin of the arrhythmia.

Our optical mapping results provide elaborated insight into discordant alternans and how it leads to increased vulnerability to re-entrant arrhythmias. Only during β-adrenergic stimulation in RyR2^A690E+/− hearts, we found spatial homogeneities in cardiac tissue displayed spatially discordant alternans, which favour re-entry, triggering ectopic beats, and facilitating the onset of lethal arrhythmic events. We suggest that discordant alternans could occur without spatial inhomogeneities of cardiac tissue properties because of tissue heterogeneity involving complex interaction of APD and CV with rating heart beats response to β-adrenergic stimulation.⁴⁵ The spatially discordant phenomenon induced by the combined action of RyR2 and β-adrenergic stimulation is consistent with the catecholamine-sensitive arrhythmia phenotype.

In summary, we identified a new mutation as a novel CPVT-causing mutation. Our data implicate that the A690E mutation causes a hypersensitive leaky RyR2 channel, responsible for aberrant Ca²⁺ handling. The abnormal spontaneous Ca²⁺ leak during diastole together with an imbalance of Ca²⁺ cycling dynamic is responsible for CaT alternans at high pacing rates, simulating the human VT phenotype underlying fast heart rate. This study illuminates the molecular and cellular pathogenesis of a novel CPVT-causing mutation, which may have important implications for future development of new therapeutic strategies.

Supplementary material

Supplementary material is available at Europace online.

Funding

This work was supported by grants from the National Natural Science Foundation of China (82070329 and 81870246 to Z.L., 81800303 to D.Z., and 81900292 to J.X.), the Shanghai Sailing Program (19YF1438000 to J.X.), the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (ZYYCXTD-C-202207 to P.L.), and the Science and Technology Department of Sichuan Province (2022YFS0613 to D.Z. and 2022YFS0618 to P.L.).

Data availability

All relevant data are within the manuscript and its Supplementary material online files.

Translational perspective

There is no effective treatment for catecholaminergic polymorphic ventricular tachycardia (CPVT); implantable cardiac defibrillators serve as a ‘back up’ strategy for high risk patients. Our research conducted a fully integrated assessment on a novel point mutation related to a single amino acid substitution (A690E) in ryanodine receptor 2 (RYR2) gene, identified from a patient with family inheritance of CPVT. The mutation is located directly at the FK506 binding protein (FKBP)-binding domain of RyR2, known as ‘controversial-spot’ for mutations that associated with CPVT. We have characterized this RyR2 mutation both in vitro and in vivo, revealed a novel mechanism by which RyR2 channel functions are impaired via altering the interaction between RyR2 and FKBP, produced hypersensitive channels, facilitated abnormal Ca²⁺ leak from the sarcoplasmic reticulum, and responsible for Ca²⁺ alternans occurrence, which are associated with CPVT. With the confirmation of this pathogenic site, we offered a new CPVT mice model for developing and screening therapeutic drugs.

References

Author notes