In vivo trafficking and functional analysis of hERG missense variants in a CRISPR/Cas9-engineered C. elegans model of congenital long-QT syndrome

Background Loss-of-function variants in KCNH2, which encodes the voltage-gated hERG (Kv11.1) K+ channel, are responsible for congenital long-QT syndrome type 2 (LQT2), a major cause of sudden cardiac death in young individuals. Yet, most hERG missense variants remain of uncertain significance, underscoring the need for scalable functional assays. Heterologous expression systems offer high-throughput assays but lack the native molecular context for hERG biogenesis and rely on overexpression. Induced pluripotent stem cell-derived cardiomyocytes provide a human cardiomyocyte background but are limited by fetal-like electrophysiology, inter-individual genetic variability, and limited scalability. Complex transgenic animals can reproduce the LQT2 phenotype but are limited by their complexity, time requirements and ethical rules. A conserved, scalable and tractable in vivo model is needed to overcome these different limitations. UNC-103, the C. elegans Kv channel ortholog of hERG, exhibits strong conservation in sequence, structure, and key regulators of biogenesis. Methods We developed a CRISPR/Cas9-engineered Caenorhabditis elegans in vivo model of LQT2 using fluorescently tagged UNC-103 knock-in strains. To quantify the trafficking efficiency and functional impact of hERG missense variants, we established an approach combining quantitative confocal imaging with assays of C. elegans egg-laying. Results The model faithfully recapitulated the known profiles of two previously well-characterized LQT2-associated hERG missense variants (G604S and G628S) and enabled in vivo characterization of a novel variant recently identified by our group (G603S). G604S and G603S showed trafficking defects (less then 50% of UNC-103 signal at neuromuscular junctions Figure 1A top, and increased somatic retention in ALM neurons Figure 1B), whereas G628S displayed normal trafficking (Figure 1A bottom). Functionally, G604S and G628S induced severe egg-laying defects, while G603S exhibited a milder phenotypic profile (Figure 2). Conclusions Our CRISPR/Cas9-engineered C. elegans model of LQT2 provides a physiologically relevant and scalable tool for in vivo characterization of hERG missense variants. This approach bridges the gap between in vitro assays and complex transgenic animals and opens new avenues for drug screening in LQT2.