Genetic variation in GNB5 causes bradycardia by augmenting the cholinergic response via increased acetylcholine-activated potassium current (IK,ACh).

Christiaan C Veerman,Kaomei Guan,Connie R Bezzina,Arie O Verkerk,Rianne Wolswinkel,Ronald Wilders,James Milnes,Isabella Mengarelli,Charlotte D Koopman,Diane Bakker,Teun P De Boer,Jeroen Bakkers,Mariam Hababa,Shirley C Van Amersfoorth,Elisabeth M Lodder

doi:10.1242/dmm.037994

Abstract

ABSTRACTMutations in GNB5, encoding the G-protein β5 subunit (Gβ5), have recently been linked to a multisystem disorder that includes severe bradycardia. Here, we investigated the mechanism underlying bradycardia caused by the recessive p.S81L Gβ5 variant. Using CRISPR/Cas9-based targeting, we generated an isogenic series of human induced pluripotent stem cell (hiPSC) lines that were either wild type, heterozygous or homozygous for the GNB5 p.S81L variant. These were differentiated into cardiomyocytes (hiPSC-CMs) that robustly expressed the acetylcholine-activated potassium channel [I(KACh); also known as IK,ACh]. Baseline electrophysiological properties of the lines did not differ. Upon application of carbachol (CCh), homozygous p.S81L hiPSC-CMs displayed an increased acetylcholine-activated potassium current (IK,ACh) density and a more pronounced decrease of spontaneous activity as compared to wild-type and heterozygous p.S81L hiPSC-CMs, explaining the bradycardia in homozygous carriers. Application of the specific I(KACh) blocker XEN-R0703 resulted in near-complete reversal of the phenotype. Our results provide mechanistic insights and proof of principle for potential therapy in patients carrying GNB5 mutations.This article has an associated First Person interview with the first author of the paper.

Highlights

Inherited ion channel mutations are an important cause of sinoatrial node (SAN) dysfunction in the young (Verkerk and Wilders, 2015)
To evaluate the electrophysiological consequences of the G-protein β5 subunit (Gβ5)-S81L variant we introduced it into a control human induced pluripotent stem cell (hiPSC) line (Dudek et al, 2013) by means of CRISPR/Cas9 based genome editing, generating an isogenic series consisting of wild-type, heterozygous and homozygous Gβ5-S81L hiPSC lines (Fig. S1A-C)
We showed that this effect in hiPSC-CMs, as well as the effect of cholinergic-induced bradycardia in gnb5 knockout zebrafish, can be rescued by the specific IK,ACh blocker XEN-R0703

Summary

Introduction

Inherited ion channel mutations are an important cause of sinoatrial node (SAN) dysfunction in the young (Verkerk and Wilders, 2015). Acetylcholine (ACh), released from post-ganglionic parasympathetic neurons, binds to M2 muscarinic receptors on pacemaker cells and atrial myocytes, triggering activation of heterotrimeric G-proteins that dissociate into Gα-GTP and Gβγ subunits. Of central importance is the effect of the Gβγ complex on the G protein-coupled inwardly rectifying K+ (GIRK) channel, which underlies the ACh-activated K+ current (IK,ACh). This channel is predominantly expressed in pacemaker cells and atrial myocytes (Dobrzynski et al, 2001) and is a heterotetramer consisting of Kir3.1 (encoded by KCNJ3) and Kir3.4 (encoded by KCNJ5) ion channel subunits (Krapivinsky et al, 1995). The Gβγ complex activates IK,ACh (Wickman et al, 1994) which, due to its permeability for K+ ions and inwardly rectifying properties (Sakmann et al, 1983), results in membrane potential hyperpolarization and slowing of diastolic depolarization in SAN cells, thereby decreasing spontaneous activity (DiFrancesco et al, 1989)

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Results

Conclusion