Disparate molecular mechanisms in cardiac ryanodine receptor channelopathies

Figures

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  FIGURE 1. Typical CPVT electrocardiogram (A). At rest. (B) During exercise testing. Very frequent ventricular extrasystoles, biderectional ventricular couplets (arrow) and non-sustained ventricular tachycardia (double arrowheads).

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  FIGURE 2. RyR2 residues at the NTD-CSol intra- and inter-subunit interfaces. Images of the NTD-CSol intra- and inter-subunit interfaces generated using PyMol from the RyR2 closed state (5GO9) structure. (A) The locations of residue G357 that is in close apposition to residues N3865 and T3867 on the same subunit, and the G357-N3865 and G357-T3867 distances (in Å) are depicted. (B) The locations of residue A77 on one subunit that is in close apposition to residue W3890 on the neighboring subunit and the A77-W3890 distance (in Å) are depicted.

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  FIGURE 3. A77T, R169L and G357S differentially affect RyR2 N-terminus tetramerization. Chemical cross-linking assays of HEK293 cell homogenates expressing NTWT (RyR2 residues 1-906, cMyc-tagged) or pro-arrhythmic mutants, NTA77T, NTR169L, NTG357S. (A) Cell homogenates were incubated with glutaraldehyde for the indicated time points and analyzed by SDS-PAGE (6% gels) and Western blotting using AbcMyc; monomer (M) and tetramer (T) are indicated with the arrows. (B) Densitometric analysis was carried out on the bands corresponding to tetramer and monomer moieties and used to calculate tetramer formation. Data (n ≥ 8) are normalized for WT and given as mean value ±SEM; statistical analysis was carried out using one-way Anova with Dunnett’s multiple comparisons test.

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  FIGURE 4. A77T, R169L and G357S differentially affect RyR2 N-terminus interaction with C-terminus. Co-immunoprecipitation assays from HEK293 cells co-expressing NTWT or pro-arrhythmic mutants, NTA77T, NTR169L, NTG357S, together with HA-tagged RyR2-CT (residues 3529-4967). (A) HA-RyR2-CT was immunoprecipitated with AbHA from CHAPS-solubilized cell lysates and the presence of co-precipitated NTWT/NTA77T/NTR169L/NTG357S was analyzed by SDS-PAGE (6% gels) and Western blotting using AbcMyc (top). To detect immuno-isolated HA-RyR2-CT, 1/10th of IP samples was analyzed by Western blotting using AbHA (bottom). Non-immune rabbit IgG served as negative control (non-specific binding). An aliquot of HEK293 cell lysate corresponding to 1% of the amount processed in the co-IP assay was included in the gels to assess protein expression. (B) Data summary for NT specific binding (non-immune IgG IP signal subtracted from anti-HA IP signal) following densitometric analysis and normalization to each construct’s respective lysate. Data (n ≥ 8) are normalized for WT and given as mean value ±SEM; statistical analysis was carried out using one-way Anova with Dunnett’s multiple comparisons test.

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  FIGURE 5. RyR2A77T and RyR2R169L display gain-of-function channel characteristics. Single cell Ca2+ imaging using confocal laser scanning microscopy to monitor spontaneous Ca2+ release transient events. (A) Fluorimetric traces from Cal-520 loaded single HEK293 cells expressing RyR2WT, RyR2A77T, RyR2R169L or RyR2G357S showing spontaneous Ca2+ transients and Ca2+ release induced by caffeine (10 mM) at the end of each experiment. (B) Cellular Ca2+ handling properties including spontaneous Ca2+ transient amplitude and duration, number of spontaneous Ca2+ transient events, amplitude of the caffeine response (Ca2+ store content) and proportion of oscillating cells. Data (for n ≥ 119 cells from n ≥ 6 separate experiments) are normalized for WT and given as mean value ± SEM (shown in red for clarity); statistical analysis was carried out using Kruskal–Wallis test with Dunn’s multiple comparison test.

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  FIGURE 6. A hypothetical model for deregulated RyR2 in CPVT. Drawing depicting proposed altered RyR2 inter-domain interactions due to NTD gain-of-function mutations. CPVT mutations may produce hyperactive leaky RyR2 channels by disrupting the NTD-NTD inter-subunit interaction (e.g., R169L, R169Q, R176Q) or by strengthening the NTD-CSol inter-subunit interaction (e.g., A77T, R420Q).