An optogenetic arrhythmia model to study catecholaminergic polymorphic ventricular tachycardia mutations - PubMed (original) (raw)
An optogenetic arrhythmia model to study catecholaminergic polymorphic ventricular tachycardia mutations
Elisabeth Fischer et al. Sci Rep. 2017.
Abstract
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition of abnormal heart rhythm (arrhythmia), induced by physical activity or stress. Mutations in ryanodine receptor 2 (RyR2), a Ca2+ release channel located in the sarcoplasmic reticulum (SR), or calsequestrin 2 (CASQ2), a SR Ca2+ binding protein, are linked to CPVT. For specific drug development and to study distinct arrhythmias, simple models are required to implement and analyze such mutations. Here, we introduced CPVT inducing mutations into the pharynx of Caenorhabditis elegans, which we previously established as an optogenetically paced heart model. By electrophysiology and video-microscopy, we characterized mutations in csq-1 (CASQ2 homologue) and unc-68 (RyR2 homologue). csq-1 deletion impaired pharynx function and caused missed pumps during 3.7 Hz pacing. Deletion mutants of unc-68, and in particular the point mutant UNC-68(R4743C), analogous to the established human CPVT mutant RyR2(R4497C), were unable to follow 3.7 Hz pacing, with progressive defects during long stimulus trains. The pharynx either locked in pumping at half the pacing frequency or stopped pumping altogether, possibly due to UNC-68 leakiness and/or malfunctional SR Ca2+ homeostasis. Last, we could reverse this 'worm arrhythmia' by the benzothiazepine S107, establishing the nematode pharynx for studying specific CPVT mutations and for drug screening.
Conflict of interest statement
The authors declare that they have no competing interests.
Figures
Figure 1
CSQ-1 deletion and the point mutation P319S affect pharyngeal pumping. (a) Expression of pcsq-1::csq-1::CFP in BWM cells and (b) pharynx muscle of the deletion mutant csq-1(ok2672). Scale bars: 50 μm and 20 μm, as indicated. Left panel: Fluorescence micrograph, right panel: DIC image; dashed lines indicate pharynx position. Structural features of the pharynx are labeled: TB (terminal bulb), I (isthmus), C (corpus), X (grinder). (c) Swimming cycles/min and (d) pump rate on food, of csq-1(ok2672) deletion mutants, as well as full length pcsq-1::csq-1::CFP rescue in pmyo-2::ChR2::mCherry background. Also analyzed (d) is the point mutation CSQ-1(P319S), compared to wt (number of animals tested is indicated at the base of each bar). (e) Number of pumps evoked at 3.7 Hz pulse frequency was counted for 100 light stimuli and averaged in 10 stimuli bins for the indicated number of animals. Original kymographs are depicted for wt (black), csq-1(ok2672) (red) and csq-1(gk876502) (blue; P319S). (f) Percentage of light stimuli inducing a pump across 100 light stimuli (data in e). (g) Percentage of pumps that were induced with a deviation of > 50 ms following a light stimulus (here defined as ‘jitter’). Shown (f and g) are wt (white), csq-1(ok2672) deletion mutant (grey) and P319S mutant (black). (h) Pump rate distribution (%) at 3.7 Hz pacing (white: > 4.5 Hz, blue: 3.3–4.5 Hz, green: 2.5–3.3 Hz, red: 1.6–2.5 Hz, grey: 0.5–1.6 Hz, black: 0 Hz) in the indicated csq-1 mutants, compared to wild type (wt) (n = 11–15). (i) Original EPG recordings of cut-head preparations and (j) mean of maximal pump rate of deletion and P319S mutant achieved in a stress test (470 nm, 10 ms pulses at stepwise increasing pulse rate (1 Hz steps, 5 s each step, 1–7 Hz), as indicated by blue tick marks) compared to wt (n = 10–23). Statistically significant differences, 1-way ANOVA and Bonferroni post-hoc test: ***p < 0.001; **p < 0.01; *p < 0.05.
Figure 2
Introduction of CPVT point mutations R4743C and R2729S into the C. elegans RyR2 (UNC-68). (a) Expression of punc-68::unc-68(exon1–4)::CFP in pharynx and BWM of deletion mutant unc-68(r1162). Dashed lines indicate pharynx position and features, as in Fig. 1a, arrows indicate BWM cells. (b) Pump rate on food of unc-68(r1162) and unc-68(e540) deletion mutants, compared to wt, IP3 receptor (itr-1(s73)) and UNC-13 synaptic vesicle priming factor (unc-13(s69)) reduction of function mutants (n = 9–14). (c) Stimulation of pumping by 1 µM serotonin (5-HT), before (white bars) and after activation of wt UNC-68 by application of 1 mM caffeine (grey bars). Shown is the pump rate (Hz) deduced from EPG recordings of cut-head preparations compared to unc-68 deletion mutants r1162 and e540 (n = 6–9; paired t-Test). (d,e) Pump rate on food (Hz) of unc-68(r1162) deletion mutant, its rescue with a fosmid encoding the wt genomic unc-68 locus as well as the R4743C (n = 11–21) and (e) R2729S point mutant engineered fosmids, compared to wt (n = 11–19). (f,g) Mean and SEM of swimming cycles/min of deletion mutant unc-68(r1162), rescue with wt fosmid and the R4743C point mutant fosmid, as well as the R2729S point mutant fosmid (g) compared to wt (number of animals as indicated). Statistically significant differences, 1-way ANOVA and Bonferroni post-hoc test: ***p < 0.001; **p < 0.01; *p < 0.05.
Figure 3
The R4743C mutation, analogous to human CPVT mutation R4497C, induces arrhythmia in the pharynx. (a) Original EPG recordings of intact, _pmyo-2::ChR2::mCherry-_expressing animals containing unc-68 deletion mutant r1162, its rescue with wt unc-68 fosmid, the R4743C or R2729S mutant fosmids, and wt. (b) Mean and SEM number of pumps achieved over 100 consecutive light stimuli (3.7 Hz, 35 ms), binned per 10 light pulses, as in Fig. 1e, but obtained by EPG recordings. Genotypes of the animals tested are indicated. (c) Percentage of successful light pulses followed by a pump, across all 100 light stimuli, as recorded in (b). (d,e) Analyses analogous to (b) and (c), but obtained by kymographic analysis of video recordings in intact _pmyo-2::ChR2::mCherry-_expressing animals. Statistically significant differences, 1-way ANOVA and Bonferroni post-hoc test: ***p < 0.001; **p < 0.01; *p < 0.05.
Figure 4
The benzothiazepine S107 reverses arrhythmia in the UNC-68(R4743C) mutant. (a) Original kymographs of video-microscopic recordings of the unc-68(r1162) rescue with wt and R4743C mutant fosmids, with or without incubation in 50 µM of the benzothiazepine S107 (inset) in 0.1% DMSO. (b) Mean and SEM number of pumps achieved over 100 consecutive light stimuli (3.7 Hz, 35 ms), binned per 10 light pulses, obtained by kymographic video analysis of intact, _pmyo-2::ChR2::mCherry-_expressing animals. As indicated, unc-68(r1162) deletion mutants, after 30 min incubation in 50 µM S107 (red) or without S107 (0.1% DMSO; black), were compared to wt with (green) or without (blue) S107 incubation (n = 4–9). (c) Analysis as in (b), but for r1162 deletion mutants expressing the R4743C fosmid, with S107 (green) or without (blue), or the rescue wt fosmid (red and black, respectively) (n = 20–29). (d) Percentage of successful light pulses followed by a pump, across all 100 light stimuli, as recorded in (b) and (c). Statistically significant differences, 1-way ANOVA and Bonferroni post-hoc test: ***p < 0.001; **p < 0.01; *p < 0.05.
References
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases
Research Materials
Miscellaneous