An Optogenetic Arrhythmia Model—Insertion of Several Catecholaminergic Polymorphic Ventricular Tachycardia Mutations Into Caenorhabditis elegans UNC-68 Disturbs Calstabin-Mediated Stabilization of the Ryanodine Receptor Homolog

Figures

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  FIGURE 1. Effect of catecholaminergic polymorphic ventricular tachycardia (CPVT) alleles and fkb-2 deletion on pharyngeal muscle cells (PMCs) and body-wall muscles (BWMs). (A) Unc-68(S2378L) shows a reduced normalized pump rate compared to the respective wild type (wt) control strain (young adult animals). (B) Deletion of fkb-2 [fkb-2(ok3007)] has no/minor effect on spontaneous pump rate on food. (C) Alleles R414Q and Q4623R show a reduction of normalized swimming cycles. (D) No significant effect of fkb-2 deletion on numbers of swimming cycles in comparison to the respective wild-type control. Whereas fkb-2 deletion in the pmyo-2::ChR2 background shows an increased number of swimming cycles in comparison to pmyo-2::ChR2. Bar graphs display mean values ± SEM. One-way ANOVA with the Bonferroni post-hoc test (B,D) or two-sample t-test for the comparison of only two mean values (A,C): *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

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  FIGURE 2. Pumping ability of CPVT mutation bearing strains upon 4 Hz stimulation. (A,B) In ScreenChip™ recordings, S2378L, P2460S, and Q4623R show a reduced pump rate compared to wild type in pmyo-2::ChR2 background. Box plot (25%, 75%; whisker: 1.5 IQR) includes every data point (rhomb), mean (circle), and median (line). Mean values, N-numbers, and significance levels (asterisks) are next to the corresponding box. Two-sample t-test: n.s., not significant, *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

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  FIGURE 3. Stress test (1–7 Hz) to determine the maximal pump rate of CPVT mutation-bearing strains. (A) Original recordings of electropharyngeograms (EPGs) obtained by electrophysiological characterization of cut-head preparations. Application of 1 Hz blue light pulses (1.5 mW/mm2, 35 ms) for 15 s followed by a stepwise increase (every 5 s) from 2 to 7 Hz. (B,C) unc-68(Q4623R) shows a reduced maximal pump rate in comparison to wt. Since pharyngeal pump ability sometimes varies among different experimental days, likely due to experimental conditions (room temperature, light-sensitivity of ATR), mutant alleles were always compared to the control animals recorded in the same batch of experiments. Box plots include every data point (rhomb), mean (open circle), and median (line). N-numbers, mean values, and significance levels (asterisks), referring to respective wt controls, are located either below or above the corresponding boxes. One-way ANOVA with the Bonferroni post-hoc test was performed: n.s., not significant, and ***P ≤ 0.001.

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  FIGURE 4. Effect of the benzothiazepine S107. In ScreenChip™ recordings of EPGs under blue light stimulation (4 Hz, 50 ms), the effect of S107 (50 μM, 30 min pre-incubation, shaded boxes) in comparison to the vehicle DMSO (0.1% in M9 buffer) on pump rate of UNC-68 mutants S2378L (A), P2460S (B) and Q4623R (C) was determined. Alleles have been crossed with a fkb-2 deletion [fkb-2(ok3007), violet boxes] to check for drug-induced UNC-68 stabilization via FKB-2. S107 rescues UNC-68 function of Q4623R and P2460S only in FKB-2 wt background. Box plots include mean (open circle) and median (line). Significance levels (asterisks), referring to respective controls (wild type, either with or without S107), are located above the corresponding boxes. Comparison of each strain with and without S107 treatment is indicated above the line pointing on the respective boxes. N-numbers as indicated. One-way ANOVA with the Bonferroni post-hoc test was performed: n.s., not significant, *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

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  FIGURE 5. RCaMP1h Ca2+ imaging in PMC of unc-68 and csq-1 alleles. (A) Increase of ΔF/F0 during stimulation (4 Hz, 50 ms, 470 nm) and (B) amplitude of ΔF/F0 during plateau phase (time period: 10-13 s). An increase of ΔF/F0 (%) upon light stimulation is only present in ChR2 and RcaMP1h co-expressing pharynxes and in the presence of the ChR2 cofactor ATR. (C) False-color representations of fluorescence intensity of ChR2 and RcaMP1h co-expressing pharynxes raised with and without ATR before blue light stimulation (0 s) and during stimulation (∼10 s). The terminal bulb of the pharynx, which is used for analysis, is labeled with a circle. (D,E) The null allele of unc-68, but not of csq-1, leads to an increase of cytosolic Ca2+ concentration. (F) Exemplary false-color representation of fluorescence intensity. Only worms that were able to follow 4 Hz stimulation have been included for analysis. Depicted is the mean of ΔF/F0 (%) without (A) or with SEM (D), time periods (∼50 ms), including unspecific peaks induced by blue light stimulation are not displayed here for a better outline. Box plots include every data point (rhomb), mean (open circle), and median (line). N-numbers and significance…

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  FIGURE 6. RCaMP1h Ca2+ imaging in PMC reveals effects of CPVT mutations inserted into unc-68 on cytosolic Ca2+ concentration. (A,B,G,H) Alleles Q4623R and S2378L show an increase of ΔF/F0 in comparison to wt animals upon light stimulation (10–13 s). (D,E) Whereas allele P2460S does not exhibit a significant change (10–13 s). (C,F,I) Exemplary false-color representation of fluorescence intensity. Only worms that were able to follow 4-Hz stimulation have been included for analysis. Depicted is the mean of ΔF/F0 (%) with SEM, time periods (∼50 ms), including unspecific peaks induced by blue light stimulation, are not displayed here for better outline (A,D,G). Box plots include every data point (rhomb), mean (open circle), and median (line). N-numbers and significance levels (asterisks) are located either below or above the corresponding boxes. T-test was performed for comparisons of the mean values during the plateau phase of the fluorescence rise (10–13 s): n.s., not significant, **P ≤ 0.01, and ***P ≤ 0.001.

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  FIGURE 7. Characterization of neutral amino acid insertion into UNC-68 luminal loop (lum. loop). (A,B) No effect on spontaneous pumping on food and reduced number of swimming cycles/min evoked by neutral luminal loop insertion. (C) Pumping ability is not reduced in the ScreenChip™ system (4 Hz stimulation, 60 s). (D) No effect on maximal pump rate of cut-head preparations in the stress test. (E) Exemplary false-color representation of fluorescence intensity. (F) Mean ± SEM of the ΔF/F0 of RCaMP1h fluorescence before and during light stimulation. (G) The amplitudes of ΔF/F0 (mean: 10–13 s) show no significant difference between wild-type and neutral luminal loop-expressing animals. Bar graphs represent mean ± SEM, box plots include data points (rhomb), mean (circle), and median (line). N-numbers and significance levels (asterisks) are displayed. One-way ANOVA with the Bonferroni test (A,B) or a two-sample t-test (C,D,G): n.s., not significant, and ***P ≤ 0.001.