Ye, Dan

Abstract Background and Aims Type 1 short QT syndrome (SQT1) is a genetic channelopathy caused by gain-of-function variants in KCNH2, resulting in shortened cardiac repolarization and QT intervals, which predispose patients to ventricular arrhythmias and sudden cardiac death. This study aimed to investigate the therapeutic efficacy of KCNH2-specific suppression-and-replacement (KCNH2-SupRep) gene therapy in a transgenic rabbit model of SQT1. Methods KCNH2-SupRep was developed by combining a KCNH2-shRNA with its corresponding shRNA-immune KCNH2-cDNA into an AAV9 vector, delivered directly into the aortic root (1 × 1010 vg/kg). Therapeutic efficacy was evaluated in vivo by electrocardiogram, ex vivo by optical mapping, and at cellular levels by patch-clamp, calcium imaging, and qPCR in ventricular cardiomyocytes (VCMs). Results In vivo, KCNH2-SupRep normalized the heart rate-corrected QT interval (QTc) in SQT1 rabbits, without affecting repolarization heterogeneity. Ex vivo, KCNH2-SupRep corrected the action potential duration (APD90) and resolved the increased apicobasal APD90 heterogeneity observed in untreated (UT)-SQT1 hearts, supporting an antiarrhythmic effect, which was further validated by reduced re-entry formation in silico. At cellular levels, KCNH2-SupRep prolonged APD90 in VCMs from SupRep-SQT1 rabbits closer to wildtype levels compared with UT- and sham-SQT1. Additionally, KCNH2-SupRep restored the cellular surrogate of the electro-mechanical window and normalized IKr in nearly 50% of VCMs, in line with a 50%–60% suppression of the mutant KCNH2 transcript. Conclusions This proof-of-concept study is the first to demonstrate the efficacy of gene therapy for SQT1 in a medium-sized animal model. KCNH2-SupRep gene therapy successfully corrected the pathologic phenotype in vivo, ex vivo, and at cellular levels in transgenic SQT1 rabbits.

Background: We identified a novel, ultra-rare missense variant (p.R534S) in DENND3 as a potential disease-susceptibility variant in a multi-generational pedigree of familial idiopathic ventricular fibrillation (IVF). DENND3 is a guanine nucleotide exchange factor and activator for small GTPase Rab proteins involved in endosome and ion channel membrane trafficking. However, the role of DENND3 in the heart and its potential involvement in cardiac arrhythmias are unknown. Objective: To functionally characterize DENND3-p.R534S and determine its impact on channel trafficking. Methods: Heterologous co-expression studies in TSA201 cells were performed with DENND3-WT or DENND3-p.R534S and ion channels: KCNQ1 (Kv7.1, IKs), KCNH2 (Kv11.1, IKr), SCN5A (Nav1.5, INa), or CACNA1C (Cav1.2, ICa) as well as Rab proteins (Rab4, Rab5, Rab6, Rab7, Rab11, Rab12). Patient-specific DENND3-p.R534S iPSC-derived cardiomyocytes (iPSC-CMs) and isogenic controls (IC) were created. Localization and expression of ion channel and Rab proteins was assessed using immunofluorescence (IF) by super resolution AiryScan microscopy. GTPase-Glo assays measured Rab protein activity. Electrophysiology studies using patch-clamp and FluoVolt were performed on iPSC-CM models. Results: IF imaging showed significant increases in membrane expression of KCNQ1 (11714±157to 21929±378, p<0.05), KCNH2 (11378±160 to 31965±544, p<0.05), , SCN5A (10339±8 to 11225±23, p<0.05), and CACNA1C (11879±83 to 13749±138, p<0.05) in DENND3-p.R534S compared to DENND3-WT expressing TSA201 cells. Rab5 distribution was altered significantly by the DENND3-p.R534S variant, with increased dispersion away from the nucleus (0.23±0.01 vs. 0.17±0.01, p<0.05) and increased proximity to the plasma membrane (0.16±0.02 vs. 0.23±0.01, p<0.05). Additionally, Rab5 activity was enhanced significantly (57% increase) by p.R534S versus WT (p<0.05). iPSC-CMs revealed similar increases in channel membrane accumulation and irregular arrhythmic activity including frequent action potential duration alternans and afterdepolarizations in 78% of p.R534S iPSC-CMs versus none in IC cells (p<0.05). Conclusion: DENND3 is a novel ion channel trafficking protein in the heart. The DENND3-p.R534S variant disrupts cardiac ion channel trafficking. These findings highlight DENND3’s role in cardiac ion channel distribution, cellular function, and potential association with arrhythmogenic risk.

Background— A 2% to 5% background rate of rare SCN5A nonsynonymous single nucleotide variants (nsSNVs) among healthy individuals confounds clinical genetic testing. Therefore, the purpose of this study was to enhance interpretation of SCN5A nsSNVs for clinical genetic testing using estimated predictive values derived from protein-topology and 7 in silico tools. Methods and Results— Seven in silico tools were used to assign pathogenic/benign status to nsSNVs from 2888 long-QT syndrome cases, 2111 Brugada syndrome cases, and 8975 controls. Estimated predictive values were determined for each tool across the entire SCN5A -encoded Na v 1.5 channel as well as for specific topographical regions. In addition, the in silico tools were assessed for their ability to correlate with cellular electrophysiology studies. In long-QT syndrome, transmembrane segments S3–S5+S6 and the DIII/DIV linker region were associated with high probability of pathogenicity. For Brugada syndrome, only the transmembrane spanning domains had a high probability of pathogenicity. Although individual tools distinguished case- and control-derived SCN5A nsSNVs, the composite use of multiple tools resulted in the greatest enhancement of interpretation. The use of the composite score allowed for enhanced interpretation for nsSNVs outside of the topological regions that intrinsically had a high probability of pathogenicity, as well as within the transmembrane spanning domains for Brugada syndrome nsSNVs. Conclusions— We have used a large case/control study to identify regions of Na v 1.5 associated with a high probability of pathogenicity. Although topology alone would leave the variants outside these identified regions in genetic purgatory, the synergistic use of multiple in silico tools may help promote or demote a variant’s pathogenic status.