Leaky Ca2+ release channel/ryanodine receptor 2 causes seizures and sudden cardiac death in mice
Stephan E. Lehnart, Marco Mongillo, Andrew Bellinger, Nicolas Lindegger, Bi-Xing Chen, William Hsueh, Steven Reiken, Anetta Wronska, Liam J. Drew, Chris W. Ward, W.J. Lederer, Robert S. Kass, Gregory Morley, Andrew R. Marks
Stephan E. Lehnart, Marco Mongillo, Andrew Bellinger, Nicolas Lindegger, Bi-Xing Chen, William Hsueh, Steven Reiken, Anetta Wronska, Liam J. Drew, Chris W. Ward, W.J. Lederer, Robert S. Kass, Gregory Morley, Andrew R. Marks
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Research Article Cardiology

Leaky Ca2+ release channel/ryanodine receptor 2 causes seizures and sudden cardiac death in mice

Abstract

The Ca2+ release channel ryanodine receptor 2 (RyR2) is required for excitation-contraction coupling in the heart and is also present in the brain. Mutations in RyR2 have been linked to exercise-induced sudden cardiac death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). CPVT-associated RyR2 mutations result in “leaky” RyR2 channels due to the decreased binding of the calstabin2 (FKBP12.6) subunit, which stabilizes the closed state of the channel. We found that mice heterozygous for the R2474S mutation in Ryr2 (Ryr2-R2474S mice) exhibited spontaneous generalized tonic-clonic seizures (which occurred in the absence of cardiac arrhythmias), exercise-induced ventricular arrhythmias, and sudden cardiac death. Treatment with a novel RyR2-specific compound (S107) that enhances the binding of calstabin2 to the mutant Ryr2-R2474S channel inhibited the channel leak and prevented cardiac arrhythmias and raised the seizure threshold. Thus, CPVT-associated mutant leaky Ryr2-R2474S channels in the brain can cause seizures in mice, independent of cardiac arrhythmias. Based on these data, we propose that CPVT is a combined neurocardiac disorder in which leaky RyR2 channels in the brain cause epilepsy, and the same leaky channels in the heart cause exercise-induced sudden cardiac death.

Authors

Stephan E. Lehnart, Marco Mongillo, Andrew Bellinger, Nicolas Lindegger, Bi-Xing Chen, William Hsueh, Steven Reiken, Anetta Wronska, Liam J. Drew, Chris W. Ward, W.J. Lederer, Robert S. Kass, Gregory Morley, Andrew R. Marks

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Figure 3

Hippocampal Ryr2RS/WT brain slices and channels exhibit burst activity, which can be inhibited by treatment with ryanodine or the RyR-stabilizing drug S107, respectively.

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Hippocampal Ryr2RS/WT brain slices and channels exhibit burst activity, ...
(A and B) Continuous confocal Ca2+ fluorescence imaging of the Ryr2RS/WT CA3 principal cell layer under control conditions (left), and seizure activity induced by low Mg2+ (0.5 mM) plus high K+ (8.5 mM) (middle) and following ryanodine (10 μM) treatment (right). Fluorescence (F) signals 1–3 in A correspond to regions of interest indicated by white circles in the CA3 layer in B. Data are representative of 3 experiments using Ryr2RS/WT hippocampal slices; dimensions are as indicated. (C) H&E histology (left) and RyR2 immunohistochemistry (right) of the hippocampal CA3 region in Ryr2RS/WT brain slices show increased RyR2 expression in the preserved principal cell layer. There were no histological abnormalities compared with WT (data not shown). (D) Representative Ryr2RS/WT single-channel traces from vesicles of isolated hippocampus from sedentary mice (left), after injection of NE (5 mg/kg twice over 3 hours; middle) or after 1 week treatment with S107 (5 mg/kg/h) followed by NE treatment (5 mg/kg twice over 3 hours; right). Po, mean open (To) and mean closed (Tc) times, closed state (c), and the fully open level (4 pA) are indicated. Thick bars above the 5-second traces indicate area shown at higher resolution in the 0.5-second traces. All-point histograms corresponding to the single-channel traces show increased numbers of partial openings (subconductance states) and overall increased activity of the brain channels from NE-treated Ryr2RS/WT mice (middle histogram). The histogram on the right shows more channels in the closed state (0 pA), consistent with the channel-stabilizing properties of the drug S107 that enhances binding of the calstabin2 subunit to the channel. (E) Average Po of WT and Ryr2RS/WT brain channels under different treatment conditions as indicated. Single-channel measurements were performed at a cis (cytosolic) Ca2+ concentration of 150 nM. *P < 0.05 versus NE-untreated; †P < 0.05, NE-treated WT versus Ryr2RS/WT. Each bar represents the average of 7–9 channels. Equivalent amounts of RyR2 were immunoprecipitated from brain homogenates with an RyR2 isoform–specific antibody followed by immunoblotting; bar graphs show the amount of PKA phosphorylation of RyR2 at Ser2808 (F) and the amount of calstabin2 bound to RyR2 (G) under the indicated conditions. Animals were treated with S107 via implantable osmotic pumps (5 mg/kg/h) for 7 days before NE stimulation. *P < 0.05 versus NE-untreated.