A Type 2 Ryanodine Receptor Variant in the Helical Domain 2 Associated with an Impairment of the Adrenergic Response

Figures

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  Figure 1. Pedigrees of two families. (a) Pedigree of Family 1; (b) Pedigree of Family 2. Squares and circles indicate males and females, respectively, and slashes indicate deceased individuals. For Family 1, only deceased and living D3291V carriers were indicated; thirty healthy genotype negative carriers were excluded. Genotypes and phenotypes are indicated with black and grey boxes. SD: Sudden death, ASD: Aborted Sudden Death.

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  Figure 2. Abnormal ECGs during ESTs in D3291V heterozygous carriers. (a) EST performed in an asymptomatic untreated subject (III.14, family 1) after genetic diagnosis at the age of 17 showing bigeminy, bi-directional doublets, and ventricular tachycardia at 125 W; (b) Polymorphic ventricular tachycardia during EST in a 44-year-old subject treated with 80 mg of nadolol (III.2, family 1). Her daughter (IV.3) was also treated with 80mg of nadolol and developed PVT; (c) Holter recording of an untreated child (IV.13, family 1) at the age of 2 with AVB and bi-directional ventricular doublets at 110 bpm.

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  Figure 3. Location of the D3291V-RyR2 variant. (a) The D3291V variant is located in the HD2 outside the 4 mutation hotspots (red double arrows) where CPVT mutations are mostly found [39,40]. (b) Sequence alignment of RyR orthologues and paralogues. The Uniprot accession numbers are the following: Homo sapiens (RyR1 P21817, RyR2 Q92736, RyR3 Q15413), Pan troglodytes (RyR2 A0A2I3RIJ4), Sus scrofa (RyR2 F1RHM3), Mus musculus (RyR2 E9Q401), Gallus gallus (RyR2 AOA1D5PAZ1), Xenopus tropicalis (RyR1 F7E4CO), Lepisosteus oculatus (RyR2 W5NIQ4, RyR1 W5NAB3, RyR3 W5N4D0), Drosophila melanogaster (RyR Q24498), Musca domestica (RyR A0A1I8MDA8), Caenorhabditis elegans (I2HAA6_CAEEL, unc-68). Sequence alignment was done at http://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 1 January 2018). The Aspartate 3291 is boxed in red. The variant N3308S (in blue) was identified in a CPVT patient [41].

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  Figure 4. Functional study of D3291V-RyR2 channel in HEK293 cells. (a) Representative traces of Ca2+ release under basal conditions and after cAMP treatment. (b) Percentages of oscillating WT-RyR2 (n = 29 dishes) and D3291V-RyR2 cells (n = 23 dishes) at different Ca2+ concentrations (0.1–1 mM) under basal conditions or challenged by 250 µM 8-bromo-cyclic AMP (cAMP). WT: black squares and dotted lines; WT + cAMP: black triangles and solid lines; D3291V: red squares and dotted lines; D3291V + cAMP: red triangles and solid lines. (c) Number of oscillations per min of WT-RyR2 (n = 300 cells) or D3291V-RyR2 cells (n = 265 cells) at different Ca2+ concentrations (0.1–2 mM) under basal conditions or challenged by 250 µM cAMP. (d) Percentages of oscillating WT-RyR2 (n = 29 dishes) (black squares) and D3291V-RyR2 (n = 23 dishes) (red circles) cells at different concentrations of Ca2+ (0.1–1 mM) challenged by 5 µM forskolin. (e) Number of oscillations per minute of WT-RyR2 (n = 300 cells) (black squares) or D3291V-RyR2 cells (n = 265 cells) (red circles) at different Ca2+ concentrations (0.1–2 mM) under basal condition or challenged by 5µM forskolin. (f) Representative Western blot of total proteins…

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  Figure 5. S2808 phosphorylation of both WT and D3291V in presence of cAMP: (a) Western blot of total proteins extracted from HEK293 cells transfected with eGFP-WT-hRyR2 or eGFP-D3291V-hRyR2 plasmids and treated or not with 250 μM 8-Bromo-cyclic AMP (cAMP) (n = 6) revealed with anti-RyR2 (A.a.) or anti-pSer2808 (A.b.). (b) Quantification of Ser2808 phosphorylation normalized to RyR2 total expression. # p < 0.05, WT + cAMP compared to D3291V + cAMP.

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  Figure 6. Dominant effect of D3291V in HL-1 cardiomyocytes: (a) Representative traces of Ca2+ release under basal conditions and after cAMP treatment. (b) Percentages of oscillating WT-RyR2 cells (n = 4 dishes) and D3291V-RyR2 cells (n = 5 dishes) at different Ca2+ concentrations (0.1, 0.5, 1 mM) under basal conditions or challenged by 250 µM cAMP. WT: black squares and dotted lines; WT + cAMP: black triangles and solid lines; D3291V: red squares and dotted lines; D3291V + cAMP: red triangles and solid lines. (c) Number of oscillations per minute of WT-RyR2 (n = 90 cells) or D3291V-RyR2 cells (n = 209 cells) at different Ca2+ concentrations (0.1, 0.5, 1 mM) under basal conditions or challenged by 250 µM cAMP. (d) Amplitude of the Ca2+ released at each spontaneous oscillation at different Ca2+ concentrations (0.1–1 mM) under basal conditions or challenged by 250 µM cAMP. (e) Time to peak at different Ca2+ concentrations (0.1–1 mM) under basal conditions or challenged by 250 µM cAMP. (f) Calcium transient duration at 50% (CTD50) at different Ca2+ concentrations (0.1–1 mM) under basal conditions or challenged by 250 µM cAMP. (g) Decay time 50% at different Ca2+ concentrations (0.1–1 mM) under…

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  Figure 7. Cytosolic and luminal Ca2+ regulation of the D3291V-RyR2 channels: (A) Single-channel recordings from WT-RyR2 (black) and D3291V-RyR2 (red) were recorded at a membrane holding potential of 0 mV at three different cytosolic free Ca2+ concentrations. Open events are upward deflections from zero current level (blue dotted line, c). The cytosolic solution contained 120 mM Tris and 250 mM HEPES (pH 7.4), and the cytosolic Ca2+ levels were adjusted with 0.2 mM BAPTA and 1 mM Dibromo-BAPTA in Cis. Trans solution contained 50 mM Ca2+ and 250 mM HEPES (pH 7.4). (B) Single-channel traces from WT-RyR2 (black) and D3291V-RyR2 (red) were recorded in symmetrical conditions of 250 mM CsCH3O3S, 20 mM HEPES, pH 7.4. In Trans, the desired luminal Ca2+ levels were adjusted with 1 mM BAPTA with the cytosolic free Ca2+ concentration set to 5 µM in Cis. Channel activity was taken at +20 mV holding potential. (C) Open probabilities of WT-RyR2 (black squares) and D3291V-RyR2 (red squares) obtained, under the same conditions as in A, and challenged with increasing free [Ca2+] in Cis. The sigmoidal curves resulted from the fitting using a Hill function: y = Vmax × xn/(kn + xn) where Vmax is the max…