Compromised repolarization reserve in a murine model of catecholaminergic polymorphic ventricular tachycardia caused by RyR2-R420Q mutation

Figures

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  Fig. 1. Increased ECG U-waves in RyR2-R420Q patients. A Resting ECG recordings from an RyR2-R420Q patient (right panel) and a genotype-negative family member control (left panel). Arrow indicates the large U-waves that occurred immediately following, and merging with, the preceding T-wave. B Resting ECG in a different R420Q patient. Method for analysing the T-wave and U-wave (shaded area) components of the TU complex by fitting the sum of a decaying exponential and Gaussian curve (green line), to digitized ECG recordings, as described in [25]. C The U-wave integral was greater in R420Q patients. Statistical analysis was conducted on the mean U-wave integral for each patient or control (open circles and bars) from the average of 1–3 cycles (individual cycle values from each patient or control are shown with different symbols beside the histogram bars). (C) unpaired t-test. N = 5 genotype negative family member controls and N = 4 R420Q patients. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 2. Pause-dependent ventricular ectopic beats (VEB) and AP lengthening in transgenic R420Q mouse hearts. A Monophasic action potential (MAP) and pseudo-ECG recording (pECG) measured simultaneously in an R420Q heart. When electrical pacing was turned off (top panel) there was a pause before sinus rhythm resumed, evident by the presence of P-waves (P). A VEB occurred during repolarization of the first sinus beat after the pause B Grey lines show the VEB in (A) overlaid with a different beat from the same heart that repolarizes normally on an expanded time scale (black lines). The VEB coincides with a premature ventricular complex on the ECG (double arrow), confirming this was not an artefact of MAP recording. C Pause-induced VEB occurred in 3/9 R420Q and 0/10 WT hearts. D Exemplar sinus rhythm MAP from a WT and R420Q heart. E Sinus rhythm MAPD90 was not different between WT and R420Q hearts. F Exemplar recording from an R420Q heart during and after burst pacing. G Exemplar MAP recordings from a WT and R420Q heart showing the first (thick line) and fifth (thin line) sinus beat following burst pacing. H MAPD90 was longer during the first post-pause beat in R420Q compared to WT hearts and…

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  Fig. 3. Altered repolarization in response to isoproterenol and premature stimulation in R420Q mouse hearts. A Exemplar MAP recordings from a WT and R420Q heart in control conditions (thick lines) and during stimulation with isoproterenol (100 nmol/L; ISO) (thin lines). In these examples, the WT MAPD at 90 % repolarization (MAPD90) was 98.4 ms in TYR and 66.0 ms in ISO, and the R420Q MAPD90 was 77.8 ms in TYR and 70.4 ms in ISO. B ISO shortened mean MAPD90 in WT but not R420Q hearts. C Conversely, ISO increased MAPD at 30 % repolarization in R420Q but not WT hearts. D S1S2 protocol involving progressively shorter S2 intervals (top) imposed at the end of steady pacing at 10 Hz. Exemplar recordings from a WT and R420Q heart in ISO when the final stimulus is at the normal 100 ms S1 cycle length (black lines) and at a shorter S2 interval of 80 ms (magenta lines). In the WT heart the shorter S2 elicits a single AP, whereas in the R420Q heart the same S2 pulse triggers EAD-like behaviour. Only a single S2 recording is shown for clarity but all hearts were subjected to all S2 intervals. E Percentage of hearts that developed arrhythmias in response to premature S2 electrical stimuli (PES) in…

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  Fig. 4. Increased prevalence of EADs and APD lengthening in isolated R420Q mouse ventricular myocytes. A Exemplar APs recorded in isolated WT and R420Q ventricular myocytes at 22 °C using whole-cell patch clamp. B APD90 was longer in R420Q myocytes during pacing at 1 Hz in normal Tyrode’s solution. C Exemplar WT and R420Q APs during a pace (1 Hz) pause (5 s) protocol (top panel) in the presence of ISO. Some R420Q cells developed EADs during steady pacing, and especially in the beat following the pause. D Proportion of WT and R420Q cells that developed EADs during a pacing-pause protocol in normal Tyrode’s and during ISO stimulation. E Exemplar confocal Ca2+ line scan recording of an EAD during steady pacing and F following the pace-pause protocol illustrated in (C) in an R420Q myocyte during ISO stimulation. Large numbers of spontaneous late Ca2+ sparks are visible coinciding with delayed recovery of the cellular average Ca2+ transient (green line) and membrane potential (magenta) shown normalized as an overlay. Note the different time scales in panels E & F. (B) n/N (cells/hearts) = 19/3 WT and 22/5 R420Q. (D) n/N (cells/hearts) as indicated below bars. (B) Unpaired t-test. (For…

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  Fig. 5. Increased frequency of late Ca2+ sparks (LCS) in isolated R420Q mouse ventricular myocytes. A Exemplar confocal Ca2+ line scan recordings. LCS during the Ca2+ transient decay were detected by automated algorithm in high-pass filtered recordings (white boxes, right panels). Event marked * is a diastolic Ca2+ spark occurring after [Ca2+] has returned to within 10 % of the resting level. B LCS frequency and C the full-duration at half maximum (FDHM) of the Ca2+ transient were increased in R420Q cells. D Ca2+ transient amplitude and E electrically-evoked Ca2+ release synchrony were similar between groups. F Increased LCS frequency during isoproterenol (ISO) stimulation. G LCS frequency and H Ca2+ transient duration was greater in R420Q myocytes during ISO stimulation, whereas I Ca2+ transient amplitude and J Ca2+ release synchrony were not different. (B-E) n/N = 20/4 WT and 17/6 R420Q cells/hearts, (G-J) n/N = 21/4 WT and 18/5 R420Q cells/hearts. Data points represent the mean value from each cell. Nested unpaired t-test.

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  Fig. 6. Time and voltage dependence of LCS in WT and R420Q mouse ventricular myocytes. The WT and R420Q exemplar APs from Fig. 4A were used as canonical Control and CPVT command waveforms for AP voltage clamp experiments, respectively. A Exemplar confocal Ca2+ line scan recordings in a WT (top panels) and R420Q cell (bottom panels) under voltage clamp with a Control (left panels) or CPVT AP (right panels) following standardized Ca2+ loading with a sequence of square pulses to +10 mV (see Supplementary Materials). B LCS frequency was greater in R420Q compared to WT cells under voltage clamp with a Control AP. C The frequency of LCS was not dependent on the particular AP waveform in either WT or R420Q cells (shown as the difference in LCS frequency clamped with a CPVT AP minus frequency with a Control AP). D (from top to bottom) Exemplar Ca2+ line scan recording, detected Ca2+ sparks, membrane potential, cellular average Ca2+ transient, and histogram of detected Ca2+ sparks in an R420Q cell under current clamp. Ca2+ spark events are marked by short lines on Vm and Ca2+ transient plots. Events between the peak and 90 % recovery of Ca2+ transient were included in LCS analysis, and later…

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  Fig. 7. Reduced Ca2+ release refractoriness increases LCS probability in R420Q mouse ventricular myocytes. A Exemplar line scan recordings showing pairs of LCS occurring from the same location ~70 ms apart. Middle panels show high-pass filtered recordings. Lower panels show the mean fluorescence through the centre of LCS. B Probability density of intervals between LCS pairs. Dashed lines show the median inter-LCS interval for WT (172 ms) and R420Q cells (99 ms). C Recovery of the second LCS (LCS2) relative to the first (LCS1) was slower in WT compared to D R420Q cells. (C,D) Data points represent one pair of LCS. Shaded areas indicate 95 % prediction interval. Solid lines show a single exponential fit to the data. (B,C,D) 53 LCS pairs from 6/2 WT cells/hearts and 94 LCS pairs from 3/3 R420Q myocytes/hearts.

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  Fig. 8. Hyperactive RyR2 induces CPVT-like aberrant Ca2+ dynamics in a computer model of spatially distributed Ca2+ release with rabbit electrophysiology. [42] A Simulated Ca2+ line scans with normal (Control) and hyperactive CPVT RyR2 properties B (increased Ca2+ sensitivity and reduced refractoriness). B Heatmap showing interaction between RyR2 Ca2+ sensitivity and refractoriness on LCS frequency. White and black asterisks indicate values used for Control and CPVT RyR2 simulations, respectively. C Ca2+ transient amplitude was decreased and the Ca2+ transient and AP duration was increased in CPVT (red lines) compared to Control (black lines). Inward INCX and late ICa were increased during repolarization in CPVT. D & E Amplitude recovery of pairs of LCS during simulated voltage clamp at −40 mV. F Probability density of intervals between LCS pairs, with the median interval in Control (167 ms) and CPVT (89 ms) indicated. G (upper panel) Control and CPVT simulations were paced to steady state at 1 Hz then SR Ca2+ release was blocked at the time indicated. (lower panel) Expanded time view of the final AP during RyR2 block (dashed lines). Also see Fig. S13. H A pace-pause protocol induced…