Rationale: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive RyR2 (cardiac ryanodine receptor) mediated calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide’s efficacy in CPVT. Objective: To determine whether RyR2 block independently contributes to flecainide’s efficacy for suppressing spontaneous sarcoplasmic reticulum Ca release and for preventing ventricular tachycardia in vivo. Methods and Results: We synthesized N-methylated flecainide analogues (QX-flecainide and N -methyl flecainide) and showed that N -methylation reduces flecainide’s inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N -methylation did not alter flecainide’s inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a Casq2 (cardiac calsequestrin) knockout (Casq2−/−) CPVT mouse model. In membrane-permeabilized Casq2−/− cardiomyocytes—lacking intact sarcolemma and devoid of sodium channel contribution—flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2−/− cardiomyocytes pretreated with tetrodotoxin to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous sarcoplasmic reticulum Ca release, while QX-flecainide and N -methyl flecainide did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2−/− mice, whereas N -methyl flecainide had no significant effect on arrhythmia burden, despite comparable sodium channel block. Conclusions: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.

Loss-of-function (LOF) variants in the K_V11.1 potassium channel cause long QT syndrome (LQTS). Most variants disrupt intracellular channel transport (trafficking) to the cell membrane. Since some channel inhibitors improve trafficking of K_V11.1 variants, a high-throughput screening (HTS) assay to detect trafficking enhancement would be valuable to the identification of drug candidates. The thallium (Tl⁺) flux assay technique, widely used for drug screening, was optimized using human embryonic kidney (HEK-293) cells expressing a trafficking-deficient K_V11.1 variant in 384-well plates. Assay quality was assessed using Z prime (Z’) scores comparing vehicle to E-4031, a drug that increases K_V11.1 membrane trafficking. The optimized assay was validated by immunoblot, electrophysiology experiments, and a pilot drug screen. The combination of: 1) truncating the trafficking-deficient variant K_V11.1-G601S (K_V11.1-G601S-G965*X) with the addition of 2) K_V11.1 channel activator (VU0405601) and 3) cesium (Cs⁺) to the Tl⁺ flux assay buffer resulted in an outstanding Z’ of 0.83. To validate the optimized trafficking assay, we carried out a pilot screen that identified three drugs (ibutilide, azaperone, and azelastine) that increase K_V11.1 trafficking. The new assay exhibited 100% sensitivity and specificity. Immunoblot and voltage-clamp experiments confirmed that all three drugs identified by the new assay improved membrane trafficking of two additional LQTS K_V11.1 variants. We report two new ways to increase target-specific activity in trafficking assays—genetic modification and channel activation—that yielded a novel HTS assay for identifying drugs that improve membrane expression of pathogenic K_V11.1 variants. SIGNIFICANCE STATEMENT This manuscript reports the development of a high-throughput assay (thallium flux) to identify drugs that can increase function in K_V11.1 variants that are trafficking-deficient. Two key aspects that improved the resolving power of the assay and could be transferable to other ion channel trafficking–related assays include genetic modification and channel activation.

Genetic diseases such as ion channelopathies substantially burden human health. Existing treatments are limited and not genotype specific. Here, we report a 2-step high-throughput approach to rapidly identify drug candidates for repurposing as genotype-specific therapy. We first screened 1,680 medicines using a thallium-flux trafficking assay against Kv11.1 gene variants causing long QT syndrome (LQTS), an ion channelopathy associated with fatal cardiac arrhythmia. We identified evacetrapib as a suitable drug candidate that improves membrane trafficking and activates channels. We then used deep mutational scanning to prospectively identify all Kv11.1 missense variants in an LQTS hotspot region responsive to treatment with evacetrapib. Combining high-throughput drug screens with deep mutational scanning establishes a paradigm for mutation-specific drug discovery translatable to personalized treatment of carriers with rare genetic disorders.

Polygenic scores (PGS) summarize the combined effects of common single-nucleotide polymorphisms and contribute to predictions of disease severity, but biological consequences linked to these common variants remain poorly defined. Here, we focused on polygenic liability for a measurable electrophysiologic trait (the QT interval). Prolonged QT interval, measured on patient electrocardiograms, is associated with an increased risk for cardiac arrhythmia. We investigated human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) from donors with extreme PGS (i.e., high and low) related to QT interval duration. We paired global proteomics with multiplexed affinity purification mass spectrometry (AP-MS) centered on Kv11.1 (hERG), a major determinant of QT-interval repolarization. Global proteomics indicated increased mitochondrial protein abundance in high-PGS cardiomyocytes, but this did not explain the Kv11.1 interactome. In high-PGS cells, Kv11.1 showed increased associations with myosin motor proteins and endosomal recycling machinery, consistent with altered (and potentially increased) recycling/trafficking dynamics rather than trafficking deficiency observed with most pathogenic Kv11.1 variants. This proof-of-concept study underscores a framework for linking polygenic factors to tractable biological consequences by combining patient-specific hiPSCs, proteomics and affinity-purification. Linking polygenic scores to changes in protein networks provides testable mechanisms that can be applied across many diseases. Significance Statement: Polygenic scores (PGS) predict disease risk, but how biological pathways are influenced by these common variants remains difficult to define. We generated human induced pluripotent stem cells from individuals with extreme high- and low-PGS for QT interval, a key electrocardiographic measure linked to arrhythmia risk. By combining global proteomics and interactomics for a common ion channel involved in regulating the QT interval (Kv11.1) we found mechanisms that are influenced by common genetic traits in patients. Our work connects polygenic scores to pathway-level molecular mechanisms in human cells and provides a general framework for uncovering how complex genetic architecture drives disease-relevant biology. Classification: Biological Sciences; Medical Sciences

Introduction: The voltage gated potassium ion channel K _V 11.1 plays a critical role in cardiac repolarization. Genetic variants that render Kv11.1 dysfunctional cause Long QT Syndrome (LQTS), which is associated with fatal arrhythmias. Approximately 90% of LQTS-associated variants cause intracellular protein transport (trafficking) dysfunction, which can be rescued by pharmacological chaperones like E-4031. Protein folding and trafficking decisions are regulated by chaperones, protein quality control factors, and trafficking machinery, comprising the cellular proteostasis network. Here, we test whether trafficking dysfunction is associated with alterations in the proteostasis network of pathogenic Kv11.1 variants, and whether pharmacological chaperones can normalize the proteostasis network of responsive variants. Methods: We used affinity-purification coupled with tandem mass tag-based quantitative mass spectrometry to assess protein interaction changes in human embryonic kidney (HEK293) cells expressing wild-type (WT) K _V 11.1 or trafficking-deficient channel variants in the presence or absence of E-4031. Resultsa: We identified 573 core K _V 11.1 protein interactors. Both variants K _V 11.1-G601S and K _V 11.1-G601S-G965* had significantly increased interactions with proteins responsible for folding, trafficking, and degradation compared to WT. We found that proteasomal degradation is a key component for K _V 11.1 degradation and that the K _V 11.1-G601S-G965* variant was more responsive to E-4031 treatment. This suggests a role in the C-terminal domain and the ER retention motif of K _V 11.1 in regulating trafficking. Conclusion: Our report characterizes the proteostasis network of K _V 11.1, two trafficking deficient K _V 11.1 variants, and variants treated with a pharmacological chaperone. The identified protein interactions could be targeted therapeutically to improve K _V 11.1 trafficking and treat Long QT Syndrome.