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

  • Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disturbance of the heart rhythm (arrhythmia) that is induced by stress or that occurs during exercise. Most mutations that have been linked to CPVT are found in two genes, i.e., ryanodine receptor 2 (RyR2) and calsequestrin 2 (CASQ2), two proteins fundamentally involved in the regulation of intracellular Ca2+ in cardiac myocytes. We inserted six CPVT-causing mutations via clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 into unc-68 and csq-1, the Caenorhabditis elegans homologs of RyR and CASQ, respectively. We characterized those mutations via video-microscopy, electrophysiology, and calcium imaging in our previously established optogenetic arrhythmia model. In this study, we additionally enabled high(er) throughput recordings of intact animals by combining optogenetic stimulation with a microfluidic chip system. Whereas only minor/no pump deficiency of the pharynx was observed at baseline, three mutations of UNC-68 (S2378L, P2460S, Q4623R; RyR2-S2246L, -P2328S, -Q4201R) reduced the ability of the organ to follow 4 Hz optogenetic stimulation. One mutation (Q4623R) was accompanied by a strong reduction of maximal pump rate. In addition, S2378L and Q4623R evoked an altered calcium handling during optogenetic stimulation. The 1,4-benzothiazepine S107, which is suggested to stabilize RyR2 channels by enhancing the binding of calstabin2, reversed the reduction of pumping ability in a mutation-specific fashion. However, this depends on the presence of FKB-2, a C. elegans calstabin2 homolog, indicating the involvement of calstabin2 in the disease-causing mechanisms of the respective mutations. In conclusion, we showed for three CPVT-like mutations in C. elegans RyR a reduced pumping ability upon light stimulation, i.e., an arrhythmia-like phenotype, that can be reversed in two cases by the benzothiazepine S107 and that depends on stabilization via FKB-2. The genetically amenable nematode in combination with optogenetics and high(er) throughput recordings is a promising straightforward system for the investigation of RyR mutations and the selection of mutation-specific drugs.
Author:Marcial Alexander Engel, Yves René Wörmann, Hanna Kaestner, Christina SchülerORCiDGND
Parent Title (English):Frontiers in physiology
Publisher:Frontiers Research Foundation
Place of publication:Lausanne
Document Type:Article
Date of Publication (online):2022/03/25
Date of first Publication:2022/03/25
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2023/05/22
Tag:C. elegans; CPVT; Calstabin2/FKBP12.6; RyR2; S107; arrhythmia model; calsequestrin; optogenetics
Issue:art. 691829
Article Number:691829
Page Number:18
First Page:1
Last Page:18
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
This research was funded by the Deutsche Forschungsgemeinschaft (DFG) grant SCHU3177/1-1/2 to CS. Publication was funded by Goethe University.
Institutes:Biochemie, Chemie und Pharmazie
Fachübergreifende Einrichtungen / Buchmann Institut für Molekulare Lebenswissenschaften (BMLS)
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Licence (German):License LogoCreative Commons - CC BY - Namensnennung 4.0 International