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

Figures

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  Figure 1. Family pedigree, clinical phenotype, and genotyping of a CPVT patient. (A) Genetic pedigree of study family. The catecholaminergic polymorphic ventricular tachycardia (CPVT) proband is identified by an arrow, and the A690E mutation in ryanodine receptor 2 (RyR2) gene is denoted by a filled symbol. The son of the proband (IV-1) was suspected of sudden cardiac death (SCD) at the age of 18 during a college basketball game. One of her sisters (III-6) experienced syncope and passed away in her mid-30 s while chasing a bus during rush hour; her cause of death was also SCD. Her grandparents were an intermarriage between cousins (I-1 and I-2). Her father was diagnosed with dilated cardiomyopathy (DCM), coronary heart disease (CHD), and hypertension (HT) and died in his mid-50 s. Another sister (III-4) also has CHD and HT. (B) Premature ventricular contractions (PVCs) and short episodes of polymorphic ventricular tachycardias (PVTs) were recorded in a 24 h ambulatory electrocardiogram (ECG) monitoring. (C) Mutations (highlighted) are identified in the proband, her sister (yellow columns), and health controls. Only the mutation A690E in the RYR2 gene (highlighted in red boxes) is uniquely…

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  Figure 2. A690E knock-in mice recapitulate the catecholaminergic polymorphic ventricular tachycardia (CPVT) phenotypes. (A) Kaplan–Meier survival curves of wild-type (WT) and RyR2A690E+/− mice during a 200 day period. (B) Survival rate of WT and RyR2A690E+/− mice in a pharmacological challenge test. (C) Representative traces of various types of ventricular ectopic beats of WT and RyR2A690E+/− mice in the test, (D) quantification of QTc, and (E) PR interval. Data are presented as means ± standard error of the mean (SEM). The two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test was conducted. ** P < 0.01. (F) Statistical non-sustained arrhythmias; (G) sustained arrhythmias (H), ventricular tachycardia (VT) duration of electrocardiograms (ECGs) analyses. (I) Representative traces of various types of ventricular ectopic beats in an exercise challenge test, (J) quantification of premature ventricular contraction (PVC)/h, and (K) heart rate analyses in the test. Data are presented as means ± SEM, analysed by a Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. RyR, ryanodine receptor.

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  Figure 3. In situ optical mapping of voltage and Ca2+ in hearts. (A) Isochrone maps of epicardial activation during heart apex pacing at cycle length (CL) 100 ms. (B) Quantification of conduction velocity (CV). (C) Representative optical colour APD80 map and corresponding traces with superimposition traces of 1, 2, 3, and 4 spots under sinus rhythm. (D) Quantification of average APD80. (E) APD80 heterogeneity: relatively APD80 coefficient of variation. (F) Representative optical colour CaTD80 map and corresponding traces with superimposition traces of 1, 2, 3, and 4 spots under sinus rhythm. (G) Quantification of average CaTD80. (H) CaTD80 heterogeneity: relatively CaTD80 coefficient of variation. Data are presented as means ± standard error of the mean (SEM) for n = 6 mice/group, analysed by the two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. *P < 0.05; ***P < 0.001. CaT, calcium transient.

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  Figure 4. RyR2A690E+/− hearts are prone to generate calcium transient (CaT) alternans. (A) Representative maps of ΔCaT amplitude (even-odd) at cycle length (CL) 50 ms of wild-type (WT) + isoproterenol (ISO) group, along with corresponding example CaT traces from the location indicated with a box (1 and 2) in the maps, characterized as normal circumstances without obvious CaT alternans. (B) Representative maps of ΔCaT amplitude at CL 50 ms of RyR2A690E+/− +ISO group, characterized as concordant CaT alternans. (C) Representative maps of ΔCaT amplitude at CL 50 ms of RyR2A690E+/− +ISO group, characterized as spatially discordant CaT alternans. (D) Quantification of CaT alternans ratio for each CL. CaT alternans ratio was calculated as , (CaTLarge and CaTSmall are the amplitudes of the large and small amplitude of a pair of alternating CaT). Representative superimposed CaT traces from RyR2A690E+/− hearts (E) and WT hearts (F) at S2 intervals ranging from 90 to 50 ms (interval 8 ms). (G) Quantification of recovery ratio of CaT. (H) RyR2 refractory period for both genotypes. Data are presented as means ± standard error of the mean (SEM) for n = 6 mice/group. The two-way analysis of variance…

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  Figure 5. The dynamics of epicardial propagation of arrhythmias in RyR2A690E+/− hearts. (A) Representative calcium transient (CaT) traces of VT episode induced by an extra stimulus method, corresponding to the locations (1, 2, and 3) indicated with the box in the left black-white heart map recorded from RyR2A690E+/− hearts. (B) Zoomed view of CaT traces of consecutive beats (purple frames) were shown below. (C) The phase map for corresponding three framed beats (i, ii, and iii); the conduction direction of trigger or re-entry activities are indicated by black arrows, all starting from location #2. RyR, ryanodine receptor.

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  Figure 6. Ca2+ and electrophysiological homeostasis disorders in RyR2A690E+/− cardiomyocytes. (A) Representative calcium transients (CaTs) images and corresponding CaT traces from wild-type (WT) and RyR2A690E+/− myocytes, distinct PCTs, and Ca2+ oscillations induced by isoproterenol (ISO) are indicated with arrows, respectively. (B) Pie charts of the incidence of PCT and Ca2+ oscillations. (C) Intact cardiomyocytes were incubated in normal Tyrode solution, Vm was measured using patch clamp, and action potentials (Aps) were induced by stimulator at 1 Hz. Representative traces of APs from WT and RyR2A690E+/− myocytes, with early afterdepolarizations (EADs), delayed afterdepolarizations (DADs), and trigger activities (TAs) indicated with arrows. (D–F) Their respective percentages in total tested cells. Data are presented as means ± standard error of the mean (SEM), analysed by χ2 (and Fisher's exact) test for proportions. *P < 0.05; **P < 0.01; ***P < 0.001. RyR, ryanodine receptor.

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  Figure 7. A690E mutation contributes instability of ryanodine receptor 2 (RyR2) channel. (A) Confocal line-scan images and corresponding (B) three-dimensional surface plots of Ca2+ sparks recorded in cardiomyocytes. (C) Histogram of mean frequency of Ca2+ sparks. (D) Box plot of amplitude; (E) full width at half maximum (FWHM); and (F) full duration at half maximum (FDHM) of Ca2+ sparks. Data are presented as means ± standard error of the mean (SEM), n = 25 cells from four hearts, analysed by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. *P < 0.05; and ***P < 0.001. (G) Experimental protocol and representative traces for quantification of sarcoplasmic reticulum (SR) Ca2+ leak and SR Ca2+ content. Summary data of SR Ca2+ load (H) and SR Ca2+ leak/load (I). Data are presented as means ± SEM, n = 20 cells from four hearts; analysed by Student’s t-test. ***P < 0.001. (J and K) Atomic model of human RyR2 structure is shown in grey ribbon in the cytoplasmic view and side view, respectively. FK506 binding protein (FKBP) is shown in orange ribbon. A690E mutation site is located in the SPRY1 domain (blue ribbon); other RyR2 domains around the FKBP binding site…

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  Figure 8. Rate dependent Ca2+ dynamic abnormality in RyR2A690E+/−responsible for cellular calcium transient (CaT) alternans. (A) Representative examples of Ca2+ signals and corresponding sarcomere shortening at 4 and 4.5 Hz frequencies. (B) Proportion of myocytes with Ca2+ alternans under different stimulation frequencies. (C) Threshold for cellular CaT alternans for each genotype. Each point represents the result from a single cell. Data are presented as means ± standard error of the mean (SEM). χ2 (and Fisher's exact) test for proportions, and Student’s t-test for threshold were conducted. ***P < 0.001. (D) Representative CaT trace of wild-type (WT) and RyR2A690E+/− myocytes and relative quantification of time-to-peak (E) and decay-time (F) at 1 Hz with isoproterenol (ISO) challenge. (G) Representative CaT trace of WT and RyR2A690E+/− myocytes and relative quantification of time-to-peak (H) and decay-time (I) at 4 Hz with ISO challenge. (J) Quantification of Δdiastolic Ca2+. Data are presented as means ± SEM. n = 20 cells from four hearts, analysed by Student’s t-test. ***P < 0.001. RyR, ryanodine receptor.