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Supporting Online Materials
Supplemental movie legend: Generalized
seizures
occurred
spontaneously
in
heterozygous
RyR2RS/WT mice (example shown). The video is of a 3 month old RyR2RS/WT mouse showing the characteristic progression of behavioral abnormalities
including
violent
tonic-clonic
seizures
that
occurred
repeatedly. Simultaneous recording telemetric electrocardiogram (ECG) data demonstrated sinus tachycardia without any other abnormal rhythms during the seizure activity.
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Supplemental Data RyR2 isoform-specific antibody characterization
Supplemental Figure 1 The RyR2 specific antibody (anti-RyR2; 1367-1380) was generated by immunization with the RyR2 specific peptide CKPEFNNHKDYAQEK in rabbits. Immunoblotting was performed with affinity purified RyR1 from rabbit skeletal muscle (1 µg), canine cardiac microsomes (20 µg), or mouse brain microsmes (50 µg) seperated by 6% PAGE and developed using the Odyssey system (LICOR, Inc., Lincoln, NE). Specific binding was detected in cardiac and brain microsomes, however, not to purified RyR1.
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S107 Characterization
Scheme 1 MeO
MeO
CO2 H
S S
CO2H a) NaNO2 , HCl/H2 O,
MeO H 2, Pd/C
NH2
NO2
b) Na 2S2 MeO 3
2
1
CO 2H
CO 2H
O MeO
N H
O PMe3 , Et3 N, THF, reflux
S
a) SOCl2
S
b) H2 NCH2 CH 2Cl
H N
MeO 4
LiAlH 4
Cl
NH S
Cl
5
O
MeO NH CH 2O NaBCNH3
MeO
MeO
N
CH 3
S
S 6
S107
Supplemental Figure 2: Chemical structure and synthesis of S107. Synthesis of S107 S26 (180 mg, 0.92 mmol) in MeOH (20 ml) was mixed with a 30% CH2O solution (1.5 ml, excess) and sodium cyanoborohydride (NaBCNH3) (0.4 g, excess). The reaction mixture was stirred at room temperature and the pH of the solution was
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maintained between 4 and 5 by addition of a few drop of 1N HCl. After 3 h, the solvents were removed under reduced pressure. The residue was dissolved in 20 ml ethyl acetate and washed with H2O and sat. NaHCO3 (2x10 ml). After removal of the solvents, the product S107 was purified by SiO2 column chromatography. Yield: 170 mg, 93%. The structure of S107 was confirmed by NMR, MS, and Elemental Analysis. The structure of S107 is shown in Supplemental Figure 2. Elemental Analysis of S107 - HCl salt m.p.: 222 °C decomposed; 1H-NMR (300 MHz, CD3OD): d 7.55 p.p.m. (d, 1H), 7.25 (s, 1H), 6.98 (d, 1H), 4.70 (s, broad, 2H), 3.85 (s, 3H), 3.70 (s, broad, 2H), 3.10 (s, broad 2H), 2.80 (s, 3H);
13
C-NMR (300 MHz, CD3OD): d 161.4, 136.0,
134.0, 128.5, 119.5, 115.6, 61.2, 60.0, 59.7, 37.5, 31.5; MS (m/z): [M+1]+ calculated for C11H16NOS, 210.09; found, 210.20; analysis (% calculated, % observed for C11H16ClNOS): C (53.76, 53.42), H (6.56, 6.61), N (5.70, 5.67), S (13.05, 13.49). Specificity of S107 10 µM S107 had no specific activity when tested against a panel of over 400 kinases, G-protein coupled receptors, ion channels, other enzymes, ion pumps, and other potential drug targets. The only receptor to which S107 demonstrated high affinity binding other than RyR channels was the I1 receptor which has been implicated as a target for anti-hypertensive drug treatment. Therefore, S107 was tested at a supra-pharmacologic dose in mice for anti-hypertensive activity and found to have none (data unpublished).
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Supplemental Figure 3: Brain expression patterns for RyR1, RyR2, and RyR3.
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In situ hybridization showing widespread, specific as well as partially overlapping expression of RyR1 and RyR2 in C57Bl6 adult male mouse brain. (a) Expression intensity from sagittal mouse brain sections 1.35 mm from midline reveals general expression ranking in the order RyR2 > RyR1 >> RyR3. Color coded reference map provides section orientation, specific brain regions, and boxed regions magnified in (b): the hippocampus (HIP), cerebral cortex frontal pole (CCFP), lateral hypothalamic area (LHA), and cerebellum (CE) are shown magnified in a color-coded format to document differences in expression intensity. Note that the expression intensity of RyR2 is maximal in the dentate gyrus and throughout CA1-CA3 of the hippocampal formation. Other areas of maximal RyR2 expression include the CCFP, LHA, and the olfactory bulb granular cell layer (not shown), while the cerebellum granule cell layer (CE) signal indicates moderate expression. In the cerebellum, RyR1 shows much stronger expression in the Purkinje cell layer. Images were prepared with the Allen Brain Atlas: http:www.brain-map.org; Seattle, WA: Allen Institute for Brain Science © 2006. Bars in (a) = 1 mm.
Optical Mapping of Ventricular Arrhythmias in CPVT mouse hearts Isolated WT and RyR2RS/WT Langendorff-perfused hearts were perfused with Tyrode’s solution (see Methods) at 37°C under control and ‘stress’ conditions (high-[Ca2+], 9 mM; isoproterenol, 100 nM). Optical mapping of the anterior surface of Langendorff-perfused RyR2RS/WT hearts loaded with a voltage 6
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sensitive dye (di-4-ANEPPS; Molecular Probes, Eugene, Oregon) confirmed normal epicardial activation. Under control conditions in RyR2RS/WT (Fig. 4e) and littermate WT hearts (data not shown), volume-conducted ECGs (vECGs) showed regular sinus rhythm (SR) and voltage activation mapping with simultaneous concentric breakthroughs on the anterior right and left ventricular free walls and rapid, homogeneous activation of the anterior wall within 4-5 ms consistent with earlier observations (35). However, both high-Ca2+ (9 mM) and ISO perfusion of RyR2RS/WT hearts independently resulted in sustained polymorphic VTs (Fig. 4h, 4i). During polymorphic VTs, epicardial activation patterns became highly heterogeneous including repetitive focal activation and abnormally directed voltage activation wavefronts.
Only 1 ISO perfused WT
heart showed VT under similar conditions. Our data indicate that both catecholamines or high-Ca2+ stimulation of RyR2RS/WT hearts leads to sustained rapid, polymorphic VTs that degenerate into ventricular fibrillation and are the likely mechanism of SCD in CPVT. RyR2RS/WT hearts spontaneously developed sustained VTs when perfused with high-Ca2+ or ISO but never under control conditions (n = 5/6, Fig. 4h). These data confirm our in vivo experiments and indicate a catecholaminergic and/or intracellular Ca2+ overload dependent mechanism of arrhythmia induction in CPVT.
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