The p.Arg52Leu variant in GNB2 causes a gain-of-function due to an increase of IK, ACh in stimulated hiPSC-CMs.

Abstract

Background: Initially discovered and published in 2017, the heterozygous mutation (p.Arg52Leu) in the GNB2 gene is associated with sinus node dysfunction (SND) and atrioventricular block (AVB). Electrophysiological characterization using heterologous expression systems has demonstrated that this R52L GNB2 variant results in a larger G-protein-activated K⁺ (GIRK/I_{K, ACh}) current. So far, this has not been functionally validated in patient-derived disease modelling using human induced pluripotent stem cell-derived cardiomyocytes (CM).

Methods: Here, we differentiated CMs derived from human induced pluripotent stem cells (hiPSC-CMs) of a patient carrying the pathogenic p.Arg52Leu GNB2 variant (hiPSC^R52L-CMs) and compared electrophysiological properties with those of two unrelated healthy hiPSC-CMs control lines. Retinoic acid (RA) treatment during the differentiation was used to generate hiPSC-CMs with robust acetylcholine-activated K⁺ currents (I_{K, ACh}).

Results: Patch-clamp experiments demonstrated that without GIRK channel stimulation the maximal diastolic potential (MDP) and spontaneous pacing rate did not differ between mutant and control cell lines and, moreover, a constitutively active I_{K, ACh} was not observed. Upon stimulation of GIRK channels with carbachol (CCh), hiPSC^R52L-CMs displayed an increased I_{K, ACh} density, a more hyperpolarized MDP and a slower spontaneous activity as compared to both control hiPSC-CMs cell lines, thus explaining the bradycardia reported from patients carrying the R52L GNB2 variant. Application of the specific I_{K, ACh} blocker XAF-1407 rescued the phenotype.

Conclusion: The reported, mutant GNB2 in patient-derived CMs is associated with a gain-of function upon GIRK channel stimulation and thereby a larger I_{K, ACh}, which could be potentially targeted and rescued by I_{K, ACh} blockers.

Clinical trial number: not applicable.