Dual calcium-voltage optical mapping of regional voltage and calcium signals in intact murine RyR2-R2474S hearts

Abstract

Abnormal regional variations in electrical and calcium homeostasis properties have been implicated in catecholaminergic polymorphic ventricular tachycardias (CPVT) attributable to abnormal RyR2-mediated store Ca²⁺ release, but their underlying mechanism have not been well explored in intact hearts.

Methods: We performed in vivo and ex vivo studies including high throughput mapping of Ca²⁺ transients (CaT) and transmembrane voltage (V_m) in murine wild-type (WT) and heterozygous RyR2-R2474S/+ hearts, before and during isoprenaline (ISO) challenge.

Results: ISO-challenged RyR2-R2474S/+ showed increased incidence of arrhythmia accompanied by abnormal Ca²⁺ transients compared to WT. CaT duration (CaTD) in the LV apex amongst regions studied both before and during ISO challenge in both WT and RyR2-R2474S/+ ventricles. RyR2-R2474S/+ ventricles showed prolonged CaTD, both before and during isoprenaline (ISO) challenge. Conversely, action potential durations (APD) were the same in WT and RyR2-R2474S/+ ventricles and identically reduced by ISO challenge. RyR2-R2474S/+ showed V _m-CaT latencies at time to half decay, but not rise time to peak, which were significantly prolonged compared to WT in all ventricular regions examined with ISO challenge. Following burst pacing, ventricular localized concordant alternans in CaT and APD were readily observed in RyR2-R2474S/+ but not in WT mice. Such CaT and APD alternans occurred mostly semiannually in specific regions of the ventricular pre-occurrence of VT.

Conclusion: The pro-arrhythmic RyR2-R2474S/+ phenotype in intact hearts thus directly parallels delayed regional CaT recovery properties and alteration of V _m-CaT latencies. Studies suggest that discordant localized calcium alternans are mechanistically responsible for action potential duration alternans and occurrence of VT in RyR2-R2474S/+ mice.

Keywords: Action potentials; Ca2+ transients; Catecholaminergic polymorphic ventricular tachycardia; Murine cardiac models; RyR2.

Graphical abstract

Fig. 1

ECG monitoring and optical mapping recordings in WT and RyR2-R2474S/+ mice. (A) Representative in vivo ECG recordings (i) and incidence of cardiac arrhythmia (ii) in RyR2-R2474S/+ mice before and during ISO challenge (ISO, 10 mg/kg. WT: arrhythmias in 0/6 hearts, RyR2-R2474S/+: arrhythmias in 6/6 hearts). (B) Optical recordings of responses to ISO (1 μM) challenge (i) and incidence of cardiac arrhythmia (ii) in ex vivo WT and RyR2-R2474S/+ hearts (WT: arrhythmias in 0/8 hearts, RyR2-R2474S/+: arrhythmias in 5/8 hearts). (C) Typical optical mapping recording showing pro-arrhythmic re-entry patterns in RyR2-R2474S/+ hearts but not WT hearts.

Fig. 2

Calcium transient (CaT) properties of WT and RyR2-R2474S/+ hearts. Typical (A) CaT duration (CaTD) maps at 10 Hz. (B) Averaged optical CaT traces before and during ISO challenge. (C) Averaged CaTD₅₀ (i) and CaTD₈₀ (ii) and (D) CaTD₈₀ dispersion before and during 1 μM ISO challenge. (E) Alterations in CaTD₅₀ (ΔCaTD₅₀, (i)) and CaTD₈₀ (ΔCaTD₈₀, (ii)) induced by 1 μM ISO challenge, in WT (n = 5) and RyR2-R2474S/+ hearts (n = 5). (*p < 0.05, **p < 0.01, Baseline vs. 1 μM ISO. ##p < 0.01, ###p < 0.001, WT vs RyR2-R2474S/+ mice). Two-way Analysis of Variance (ANOVA) with Sidak's test for multiple comparisons.

Fig. 3

Regional calcium transient (CaT) properties in WT and RyR2-R2474S/+ hearts. (A) Regional CaTD₅₀ and (B) CaTD₈₀ (means ± SEM) before (i) and during 1 μM ISO challenge (ii) and (C) their resulting alterations, ΔCaTD₅₀ (i) and ΔCaTD₈₀ (ii) (means ± SEM), in WT (n = 5) and RyR2-R2474S/+ hearts (n = 5). (D) Typical CaTD₈₀ heatmap and sketch map was shown for regional distribution analysis. LV = left ventricle. RV = right ventricle. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Two-way Analysis of Variance (ANOVA) with Sidak's test for multiple comparisons.

Fig. 4

Action potential properties of WT and RyR2-R2474S/+ hearts. Typical maps of action potential duration at 80 % recovery (APD₈₀) (A) and averaged optical action potential traces (B). (C) Averaged APD₅₀ (i) and APD₈₀ (ii) and (D) averaged APD₈₀ dispersion before and during 1 μM ISO challenge. (E) Alterations in APD₅₀ (ΔAPD₅₀, (i)) and APD₈₀ (ΔAPD₈₀, (ii)) induced by 1 μM ISO in WT (n = 5) and RyR2-R2474S/+ hearts (n = 5). (F) Restitution curves and ERP (G) between RyR2-R2474S/+ and WT heart with or without challenge of ISO (F) (n = 5 for WT, n = 5 for RyR2-R2474S/+ group). *p < 0.05, **p < 0.01. Two-way Analysis of Variance (ANOVA) with Sidak's test for multiple comparisons.

Fig. 5

Regional action potential properties of WT and RyR2-R2474S/+ hearts. (A) Regional APD₅₀ and (B) APD₈₀ values before (i) and during 1 μM ISO challenge (ii) and (C) the resulting ΔAPD₅₀, (i) and ΔAPD₈₀ (ii) in WT (n = 5) and RyR2-R2474S/+ hearts (n = 5). LV = left ventricle. RV = right ventricle. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Two-way Analysis of Variance (ANOVA) with Sidak's test for multiple comparisons.

Fig. 6

Comparisons of CaT and Vm findings in WT and RyR2-R2474S/+ hearts and CaT abnormalities during ISO challenge in RyR2-R2474S/+ hearts. Typical voltage‑calcium latency mapping of (A) half rise time and (B) time to 50 % full recovery and averaged dual voltage‑calcium signals. (C) Averaged time lags between their half rise time, (i), time to peak (ii) and time to half recovery (iii) before and during 1 μM ISO challenge in WT (n = 4) and RyR2-R2474S/+ hearts (n = 4). (D) frequency-dependent CaT alternans was observed in RyR2-R2474S/+ hearts after ISO challenge, not in WT mice. Changeable CaT alternans manifested as SDA and SCA (E) and phase maps showing reentrant activity (F) under 50 Hz burst pacing after ISO challenge in RyR2-R2474S/+ hearts. #p < 0.05, ##p < 0.01 WT vs. RyR2-R2474S/+ mice. Two-way Analysis of Variance (ANOVA) with Sidak's test for multiple comparisons.

Fig. 7

Regional voltage-calcium properties in WT and RyR2-R2474S/+ hearts. Regional latencies between (A) half rise time, (B) time to peak and (C) time to half recovery before (i) and following 1 μM ISO challenge (ii), and (D) the resulting changes in latencies between half rise time (i), time to peak (ii) and time to half recovery (iii), in WT (n = 4) and RyR2-R2474S/+ mice (n = 4). (E) Schematic diagram of voltage‑calcium latency. LV = left ventricle. RV = right ventricle. *p < 0.05, **p < 0.01, ***p < 0.001. Two-way Analysis of Variance (ANOVA) with Sidak's test for multiple comparisons.

Supplementary Fig. 1

Action potential conduction properties in WT and RyR2-R2474S/+ hearts. (A) Typical isochronal maps. (B) Averaged conduction velocities and (C) conduction velocity dispersion before and following 1 μM ISO challenge in WT (n = 5) and RyR2-R2474S/+ hearts (n = 5). Panels D to I are based on voltage dye-only (RH237) optical mapping at baseline. (D) typical APD₈₀ and APD₅₀ maps of WT and RyR2-R2474S/+ hearts. (E) Statistical results of APD₅₀ (i) and its dispersion (ii). (F) Statistical results of APD₈₀ (i) and its dispersion (ii). Isochronal maps (G), activation curves (H) and CV statistical results (I) of WT and RyR2-R2474S/+ hearts. (n = 5 for each group).