Bruton's tyrosine kinase inhibitor-related cardiotoxicity: The quest for predictive biomarkers and improved risk stratification.

Ibrutinib was the first Bruton's tyrosine kinase (BTK) inhibitor approved for the treatment of patients with chronic lymphocytic leukemia (CLL). While producing durable responses and prolonging survival, roughly 20-25% of patients experience dose limiting side effects, mostly consisting of cardiovascular toxicities like severe hypertension and atrial fibrillation. While clinical predictors of BTK inhibitor-related cardiotoxicity have been proposed and may aid in risk stratification, there is no routine risk model used in clinical practice today to identify patients at highest risk. A recent study investigating genetic predictors of ibrutinib-related cardiotoxicity found that single nucleotide polymorphisms in KCNQ1 and GATA4 were significantly associated with cardiotoxic events. If replicated in larger studies, these biomarkers may improve risk stratification in combination with clinical factors. A clinicogenomic risk model may aid in identifying patients at highest risk of developing BTK inhibitor-related cardiotoxicity in which further risk mitigation strategies may be explored.

Phosphoproteomics reveals a novel mechanism underlying the proarrhythmic effects of nilotinib, vandetanib, and mobocertinib.

The use of tyrosine kinase inhibitors (TKIs) has resulted in significant occurrence of arrhythmias. However, the precise mechanism of the proarrhythmic effect is not fully understood. In this study, we found that nilotinib (NIL), vandetanib (VAN), and mobocertinib (MOB) induced the development of "cellrhythmia" (arrhythmia-like events) in a concentration-dependent manner in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Continuous administration of NIL, VAN, or MOB in animals significantly prolonged the action potential durations (APD) and increased susceptibility to arrhythmias. Using phosphoproteomic analysis, we identified proteins with altered phosphorylation levels after treatment with 3 µM NIL, VAN, and MOB for 1.5 h. Using these identified proteins as substrates, we performed kinase-substrate enrichment analysis to identify the kinases driving the changes in phosphorylation levels of these proteins. MAPK and WNK were both inhibited by NIL, VAN, and MOB. A selective inhibitor of WNK1, WNK-IN-11, induced concentration- and time-dependent cellrhythmias and prolonged field potential duration (FPD) in hiPSC-CMs in vitro; furthermore, administration in guinea pigs confirmed that WNK-IN-11 prolonged ventricular repolarization and increased susceptibility to arrhythmias. Fingding indicated that WNK1 inhibition had an in vivo and in vitro arrhythmogenic phenotype similar to TKIs. Additionally,three of TKIs reduced hERG and KCNQ1 expression at protein level, not at transcription level. Similarly, the knockdown of WNK1 decreased hERG and KCNQ1 protein expression in hiPSC-CMs. Collectively, our data suggest that the proarrhythmic effects of NIL, VAN, and MOB occur through a kinase inhibition mechanism. NIL, VAN, and MOB inhibit WNK1 kinase, leading to a decrease in hERG and KCNQ1 protein expression, thereby prolonging action potential repolarization and consequently cause arrhythmias.

A mutation in the cardiac KV7.1 channel possibly disrupts interaction with Yotiao protein.

Here, we characterized the p.Arg583His (R583H) Kv7.1 mutation, identified in two unrelated families suffered from LQT syndrome. This mutation is located in the HС-HD linker of the cytoplasmic portion of the Kv7.1 channel. This linker, together with HD helix are responsible for binding the A-kinase anchoring protein 9 (AKAP9), Yotiao. We studied the electrophysiological characteristics of the mutated channel expressed in CHO-K1 along with KCNE1 subunit and Yotiao protein, using the whole-cell patch-clamp technique. We found that R583H mutation, even at the heterozygous state, impedes IKs activation. Molecular modeling showed that HС and HD helixes of the C-terminal part of Kv7.1 channel are swapped along the C-terminus length of the channel and that R583 position is exposed to the outer surface of HC-HD tandem coiled-coil. Interestingly, the adenylate cyclase activator, forskolin had a smaller effect on the mutant channel comparing with the WT protein, suggesting that R583H mutation may disrupt the interaction of the channel with the adaptor protein Yotiao and, therefore, may impair phosphorylation of the KCNQ1 channel.

Generation of a KCNQ1 (c.1032 + 2 T > C) mutant human embryonic stem cell line via CRISPR base editing.

The KCNQ1 gene encodes a voltage-gated potassium channel, which plays an important role in the repolarization of myocardial action potentials. Mutations in this gene often result in type 1 long QT syndrome (LQT1). Here, we generated a KCNQ1 (c.1032 + 2 T > C) mutant human embryonic stem cell line (WAe009-A-1D) based on the transient expression adenine base editing system that converts base A to G. The WAe009-A-1D cell maintains the morphology, pluripotency, and normal karyotype of the stem cells and is capable of differentiating into all three germ layers in vivo.

The electrophysiologic effects of KCNQ1 extend beyond expression of IKs: evidence from genetic and pharmacologic block.

AIMS: While variants in KCNQ1 are the commonest cause of the congenital long QT syndrome, we and others find only a small IKs in cardiomyocytes from human-induced pluripotent stem cells (iPSC-CMs) or human ventricular myocytes. METHODS AND RESULTS: We studied population control iPSC-CMs and iPSC-CMs from a patient with Jervell and Lange-Nielsen (JLN) syndrome due to compound heterozygous loss-of-function (LOF) KCNQ1 variants. We compared the effects of pharmacologic IKs block to those of genetic KCNQ1 ablation, using JLN cells, cells homozygous for the KCNQ1 LOF allele G643S, or siRNAs reducing KCNQ1 expression. We also studied the effects of two blockers of IKr, the other major cardiac repolarizing current, in the setting of pharmacologic or genetic ablation of KCNQ1: moxifloxacin, associated with a very low risk of drug-induced long QT, and dofetilide, a high-risk drug. In control cells, a small IKs was readily recorded but the pharmacologic IKs block produced no change in action potential duration at 90% repolarization (APD90). In contrast, in cells with genetic ablation of KCNQ1 (JLN), baseline APD90 was markedly prolonged compared with control cells (469 ± 20 vs. 310 ± 16 ms). JLN cells displayed increased sensitivity to acute IKr block: the concentration (µM) of moxifloxacin required to prolong APD90 100 msec was 237.4 [median, interquartile range (IQR) 100.6-391.6, n = 7] in population cells vs. 23.7 (17.3-28.7, n = 11) in JLN cells. In control cells, chronic moxifloxacin exposure (300 µM) mildly prolonged APD90 (10%) and increased IKs, while chronic exposure to dofetilide (5 nM) produced greater prolongation (67%) and no increase in IKs. However, in the siRNA-treated cells, moxifloxacin did not increase IKs and markedly prolonged APD90. CONCLUSION: Our data strongly suggest that KCNQ1 expression modulates baseline cardiac repolarization, and the response to IKr block, through mechanisms beyond simply generating IKs.

Generation of an induced pluripotent stem cell line from patient with atrial fibrillation with KCNQ1 p.Ser209Pro mutation.

Gain-of-function mutations in the KCNQ1 gene can cause atrial fibrillation. In this study, we generated an induced stem cell line (GRCHJUi001) from one member of an atrial fibrillation family line, whom had heterozygous mutation in the KCNQ1 gene c.625 T > C (p.Ser209Pro), and the cell line showed maintenance of stem cells characterized by morphology, normal karyotype, and pluripotency.

Correlation Analysis of KCNQ1 Gene Polymorphism with Blood Indexes and Prognosis of Non-Small Cell Lung Cancer.

BACKGROUND: This study was conducted to investigate the correlation between KCNQ1 rs2237895 A/C gene polymorphism and blood indexes and prognosis in non-small cell lung cancer (NSCLC). METHODS: A total of 260 NSCLC patients were selected and classified into stage I - II (n = 109) and stage III - IV (n = 151) according to by American Joint Committee on Cancer Staging Manual. A control group was established with another 92 healthy subjects. The genotype distribution of rs2237895 was analyzed in all subjects. 2 analysis or Fisher's test was employed to analyze the association between genotype and allele distribution frequencies with carcinoembryonic antigen (CEA), squamous cell carcinoma antigen, and cytokeratin fragment 19 (CyfrA 21-1). Overall survival was compared by genotype stratification using Kaplan-Meier analysis. Univariate and multivariate Cox risk regression analyses were used to determine the prognostic value of allele C in NSCLC. RESULTS: AC/CC genotypes in NSCLC patients were associated with gender, hypertension, smoking, clinical TNM stage, lymph node metastasis, and distant metastasis. C allele was associated with higher risk levels of serum tumor markers. Patients with allele C (AC + CC) had lower overall survival than patients with genotype AA. Finally, clinical stage, lymph node metastasis, higher CEA and CyfrA 21-1 serum levels, and rs2237895 A/C gene poly-morphism were independent prognostic factors of NSCLC. CONCLUSIONS: rs2237895 A/C polymorphism of the KCNQ1 gene can be a prognostic predictor in patients with surgically treated NSCLC.

KCNQ1 p.D446E Variant as a Risk Allele for Arrhythmogenic Phenotypes: Electrophysiological Characterization Reveals a Complex Phenotype Affecting the Slow Delayed Rectifier Potassium Current (IKs) Voltage Dependence by Causing a Hyperpolarizing Shift and a Lack of Response to Protein Kinase A Activation.

Genetic testing is crucial in inherited arrhythmogenic channelopathies; however, the clinical interpretation of genetic variants remains challenging. Incomplete penetrance, oligogenic, polygenic or multifactorial forms of channelopathies further complicate variant interpretation. We identified the KCNQ1/p.D446E variant in 2/63 patients with long QT syndrome, 30-fold more frequent than in public databases. We thus characterized the biophysical phenotypes of wildtype and mutant IKs co-expressing these alleles with the ß-subunit minK in HEK293 cells. KCNQ1 p.446E homozygosity significantly shifted IKs voltage dependence to hyperpolarizing potentials in basal conditions (gain of function) but failed to shift voltage dependence to hyperpolarizing potentials (loss of function) in the presence of 8Br-cAMP, a protein kinase A activator. Basal IKs activation kinetics did not differ among genotypes, but in response to 8Br-cAMP, IKs 446 E/E (homozygous) activation kinetics were slower at the most positive potentials. Protein modeling predicted a slower transition of the 446E Kv7.1 tetrameric channel to the stabilized open state. In conclusion, biophysical and modelling evidence shows that the KCNQ1 p.D446E variant has complex functional consequences including both gain and loss of function, suggesting a contribution to the pathogenesis of arrhythmogenic phenotypes as a functional risk allele.

Escitalopram-induced QTc prolongation and its relationship with KCNQ1, KCNE1, and KCNH2 gene polymorphisms.

BACKGROUND: Escitalopram can cause prolongation of the QT interval on the electrocardiogram (ECG). However, only some patients get pathological QTc prolongation in clinic. We investigated the influence of KCNQ1, KCNE1, and KCNH2 gene polymorphisms along with clinical factors on escitalopram-induced QTc prolongation. METHODS: A total of 713 patients prescribed escitalopram were identified and had at least one ECG recording in this retrospective study. 472 patients with two or more ECG data were divided into QTc prolongation (n = 119) and non-prolongation (n = 353) groups depending on the threshold change in QTc of 30 ms above baseline value (∆QTc ≥ 30 ms). 45 patients in the QTc prolongation group and 90 patients in the QTc non-prolongation group were genotyped for 43 single nucleotide polymorphisms (SNPs) of KCNQ1, KCNE1, and KCNH2 genes. RESULTS: Patients with QTc prolongation (∆QTc ≥ 30 ms) got higher escitalopram dose (10.3 mg) than patients without QTc prolongation (9.4 mg), although no significant relationship was found between QTc interval and escitalopram dose in the linear mixed model. Patients who were older/coronary disease/hypertension or carried with KCNE1 rs1805127 C allele, KCNE1 rs4817668 C allele, KCNH2 rs3807372 AG/GG genotype were significantly at risk for QTc prolongation (∆QTc ≥ 30 ms). Concomitant antipsychotic treatment was associated with a longer QTc interval. LIMITATIONS: A relatively small sample size and lack of the blood concentration of escitalopram restricted the accurate relationship between escitalopram dose and QTc interval. CONCLUSION: Our study revealed that KCNQ1, KCNE1, and KCNH2 gene polymorphisms along with clinical factors provide a complementary effect in escitalopram-induced QTc prolongation.

Proof of concept for monoclonal antibody therapy in a cellular model of acquired long QT syndrome type 3.

Long QT syndrome (LQTS) type 3 although less common than the first two forms, differs in that arrhythmic events are less likely triggered by adrenergic stimuli and are more often lethal. Effective pharmacological treatment is challenged by interindividual differences, mutation dependence, and adverse effects, translating into an increased use of invasive measures (implantable cardioverter-defibrillator, sympathetic denervation) in patients with LQTS type 3. Previous studies have demonstrated the therapeutic potential of polyclonal KCNQ1 antibody for LQTS type 2. Here, we sought to identify a monoclonal KCNQ1 antibody that preserves the electrophysiological properties of the polyclonal form. Using hybridoma technology, murine monoclonal antibodies were generated, and patch clamp studies were performed for functional characterization. We identified a monoclonal KCNQ1 antibody able to normalize cardiac action potential duration and to suppress arrhythmias in a pharmacological model of LQTS type 3 using human-induced pluripotent stem cell-derived cardiomyocytes.NEW & NOTEWORTHY Long QT syndrome is a leading cause of sudden cardiac death in the young. Recent research has highlighted KCNQ1 antibody therapy as a new treatment modality for long QT syndrome type 2. Here, we developed a monoclonal KCNQ1 antibody that similarly restores cardiac repolarization. Moreover, the identified monoclonal KCNQ1 antibody suppresses arrhythmias in a cellular model of long QT syndrome type 3, holding promise as a first-in-class antiarrhythmic immunotherapy.

Imprinted gene alterations in the kidneys of growth restricted offspring may be mediated by a long non-coding RNA.

Altered epigenetic mechanisms have been previously reported in growth restricted offspring whose mothers experienced environmental insults during pregnancy in both human and rodent studies. We previously reported changes in the expression of the DNA methyltransferase Dnmt3a and the imprinted genes Cdkn1c (Cyclin-dependent kinase inhibitor 1C) and Kcnq1 (Potassium voltage-gated channel subfamily Q member 1) in the kidney tissue of growth restricted rats whose mothers had uteroplacental insufficiency induced on day 18 of gestation, at both embryonic day 20 (E20) and postnatal day 1 (PN1). To determine the mechanisms responsible for changes in the expression of these imprinted genes, we investigated DNA methylation of KvDMR1, an imprinting control region (ICR) that includes the promoter of the antisense long non-coding RNA Kcnq1ot1 (Kcnq1 opposite strand/antisense transcript 1). Kcnq1ot1 expression decreased by 51% in growth restricted offspring compared to sham at PN1. Interestingly, there was a negative correlation between Kcnq1ot1 and Kcnq1 in the E20 growth restricted group (Spearman's ρ = 0.014). No correlation was observed between Kcnq1ot1 and Cdkn1c expression in either group at any time point. Additionally, there was a 11.25% decrease in the methylation level at one CpG site within KvDMR1 ICR. This study, together with others in the literature, supports that long non-coding RNAs may mediate changes seen in tissues of growth restricted offspring.

Enhancing the interpretation of genetic observations in KCNQ1 in unselected populations: relevance to secondary findings.

AIMS: Rare variants in the KCNQ1 gene are found in the healthy population to a much greater extent than the prevalence of Long QT Syndrome type 1 (LQTS1). This observation creates challenges in the interpretation of KCNQ1 rare variants that may be identified as secondary findings in whole exome sequencing.This study sought to identify missense variants within sub-domains of the KCNQ1-encoded Kv7.1 potassium channel that would be highly predictive of disease in the context of secondary findings. METHODS AND RESULTS: We established a set of KCNQ1 variants reported in over 3700 patients with diagnosed or suspected LQTS sent for clinical genetic testing and compared the domain-specific location of identified variants to those observed in an unselected population of 140 000 individuals. We identified three regions that showed a significant enrichment of KCNQ1 variants associated with LQTS at an odds ratio (OR) >2: the pore region, and the adjacent 5th (S5) and 6th (S6) transmembrane (TM) regions. An additional segment within the carboxyl terminus of Kv7.1, conserved region 2 (CR2), also showed an increased OR of disease association. Furthermore, the TM spanning S5-Pore-S6 region correlated with a significant increase in cardiac events. CONCLUSION: Rare missense variants with a clear phenotype of LQTS have a high likelihood to be present within the pore and adjacent TM segments (S5-Pore-S6) and a greater tendency to be present within CR2. This data will enhance interpretation of secondary findings within the KCNQ1 gene. Further, our data support a more severe phenotype in LQTS patients with variants within the S5-Pore-S6 region.

Pairwise biosynthesis of ion channels stabilizes excitability and mitigates arrhythmias.

Coordinated expression of ion channels is crucial for cardiac rhythms, neural signaling, and cell cycle progression. Perturbation of this balance results in many disorders including cardiac arrhythmias. Prior work revealed association of mRNAs encoding cardiac NaV1.5 (SCN5A) and hERG1 (KCNH2), but the functional significance of this association was not established. Here, we provide a more comprehensive picture of KCNH2, SCN5A, CACNA1C, and KCNQ1 transcripts collectively copurifying with nascent hERG1, NaV1.5, CaV1.2, or KCNQ1 channel proteins. Single-molecule fluorescence in situ hybridization (smFISH) combined with immunofluorescence reveals that the channel proteins are synthesized predominantly as heterotypic pairs from discrete molecules of mRNA, not as larger cotranslational complexes. Puromycin disrupted colocalization of mRNA with its encoded protein, as expected, but remarkably also pairwise mRNA association, suggesting that transcript association relies on intact translational machinery or the presence of the nascent protein. Targeted depletion of KCHN2 by specific shRNA resulted in concomitant reduction of all associated mRNAs, with a corresponding reduction in the encoded channel currents. This co-knockdown effect, originally described for KCNH2 and SCN5A, thus appears to be a general phenomenon among transcripts encoding functionally related proteins. In multielectrode array recordings, proarrhythmic behavior arose when IKr was reduced by the selective blocker dofetilide at IC50 concentrations, but not when equivalent reductions were mediated by shRNA, suggesting that co-knockdown mitigates proarrhythmic behavior expected from the selective reduction of a single channel species. We propose that coordinated, cotranslational association of functionally related ion channel mRNAs confers electrical stability by co-regulating complementary ion channels in macromolecular complexes.

A generic binding pocket for small molecule IKs activators at the extracellular inter-subunit interface of KCNQ1 and KCNE1 channel complexes.

The cardiac IKs ion channel comprises KCNQ1, calmodulin, and KCNE1 in a dodecameric complex which provides a repolarizing current reserve at higher heart rates and protects from arrhythmia syndromes that cause fainting and sudden death. Pharmacological activators of IKs are therefore of interest both scientifically and therapeutically for treatment of IKs loss-of-function disorders. One group of chemical activators are only active in the presence of the accessory KCNE1 subunit and here we investigate this phenomenon using molecular modeling techniques and mutagenesis scanning in mammalian cells. A generalized activator binding pocket is formed extracellularly by KCNE1, the domain-swapped S1 helices of one KCNQ1 subunit and the pore/turret region made up of two other KCNQ1 subunits. A few residues, including K41, A44 and Y46 in KCNE1, W323 in the KCNQ1 pore, and Y148 in the KCNQ1 S1 domain, appear critical for the binding of structurally diverse molecules, but in addition, molecular modeling studies suggest that induced fit by structurally different molecules underlies the generalized nature of the binding pocket. Activation of IKs is enhanced by stabilization of the KCNQ1-S1/KCNE1/pore complex, which ultimately slows deactivation of the current, and promotes outward current summation at higher pulse rates. Our results provide a mechanistic explanation of enhanced IKs currents by these activator compounds and provide a map for future design of more potent therapeutically useful molecules.

Autosomal Recessive Long QT Syndrome: Clinical Aspects and Therapy.

The autosomal recessive (AR) form of Long QT Syndrome (LQTS) is described both associated with deafness known as Jervell and Lange-Nielsen (JLN) syndrome, and without deafness (WD). The aim of the study is to report the characteristics of AR LQTS patients and the efficacy of the therapy. Data of all children with AR LQTS referred to the Bambino Gesù Children's Hospital IRCCS from September 2012 to September 2021were included. Three (30%) patients had compound heterozygosity and 7 (70%) had homozygous variants of the KCNQ1 gene, the latter showing deafness. Four patients (40%) presented aborted sudden cardiac death (aSCD): three with previous episodes of syncope (75%), the other without previous symptoms (16.6% of asymptomatic patients). An episode of aSCD occurred in 2/3 (66.7%) of WD and heterozygous patients, while in 2/7 (28%) JLN and homozygous patients and in 2/2 patients with QTC > 600 ms. All patients were treated with Nadolol. In 5 Mexiletine was added, shortening QTc and obtaining the disappearance of the T-wave alternance (TWA) in 3/3. Episodes of aSCD seem to be more frequent in LQTS patients with compound heterozygous variants and WD than in those with JLN and homozygous variants. Episodes of aSCD also appear more frequent in children with syncope or with QTc value > 600 ms, even on beta-blocker therapy, than in patients without syncope or with Qtc < 600 ms. However, our descriptive results should be confirmed by larger studies. Moreover, Mexiletine addition reduced QTc value and eliminated TWA.

Novel combinations of variations in KCNQ1 were associated with patients with long QT syndrome or Jervell and Lange-Nielsen syndrome.

OBJECTIVES: Long QT syndrome (LQTS) is one of the primary causes of sudden cardiac death (SCD) in youth. Studies have identified mutations in ion channel genes as key players in the pathogenesis of LQTS. However, the specific etiology in individual families remains unknown. METHODS: Three unrelated Chinese pedigrees diagnosed with LQTS or Jervell and Lange-Nielsen syndrome (JLNS) were recruited clinically. Whole exome sequencing (WES) was performed and further validated by multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing. RESULTS: All of the probands in our study experienced syncope episodes and featured typically prolonged QTc-intervals. Two probands also presented with congenital hearing loss and iron-deficiency anemia and thus were diagnosed with JLNS. A total of five different variants in KCNQ1, encoding a subunit of the voltage-gated potassium channel, were identified in 3 probands. The heterozygous variants, KCNQ1 c.749T > C was responsible for LQTS in Case 1, transmitting in an autosomal dominant pattern. Two patterns of compound heterozygous variants were responsible for JLNS, including a large deletion causing loss of the exon 16 and missense variant c.1663 C > T in Case 2, and splicing variant c.605-2 A > G and frame-shift variant c.1265del in Case 3. To our knowledge, the compound heterozygous mutations containing a large deletion and missense variant were first reported in patients with JLNS. CONCLUSION: Our study expanded the LQTS genetic spectrum, thus favoring disease screening and diagnosis, personalized treatment, and genetic consultation.

Long QT syndrome-associated calmodulin variants disrupt the activity of the slowly activating delayed rectifier potassium channel.

Calmodulin (CaM) is a highly conserved mediator of calcium (Ca2+ )-dependent signalling and modulates various cardiac ion channels. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS). LQTS patients display prolonged ventricular recovery times (QT interval), increasing their risk of incurring life-threatening arrhythmic events. Loss-of-function mutations to Kv7.1 (which drives the slow delayed rectifier potassium current, IKs, a key ventricular repolarising current) are the largest contributor to congenital LQTS (>50% of cases). CaM modulates Kv7.1 to produce a Ca2+ -sensitive IKs, but little is known about the consequences of LQTS-associated CaM mutations on Kv7.1 function. Here, we present novel data characterising the biophysical and modulatory properties of three LQTS-associated CaM variants (D95V, N97I and D131H). We showed that mutations induced structural alterations in CaM and reduced affinity for Kv7.1, when compared with wild-type (WT). Using HEK293T cells expressing Kv7.1 channel subunits (KCNQ1/KCNE1) and patch-clamp electrophysiology, we demonstrated that LQTS-associated CaM variants reduced current density at systolic Ca2+ concentrations (1 µm), revealing a direct QT-prolonging modulatory effect. Our data highlight for the first time that LQTS-associated perturbations to CaM's structure impede complex formation with Kv7.1 and subsequently result in reduced IKs. This provides a novel mechanistic insight into how the perturbed structure-function relationship of CaM variants contributes to the LQTS phenotype. KEY POINTS: Calmodulin (CaM) is a ubiquitous, highly conserved calcium (Ca2+ ) sensor playing a key role in cardiac muscle contraction. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS), a life-threatening cardiac arrhythmia syndrome. LQTS-associated CaM variants (D95V, N97I and D131H) induced structural alterations, altered binding to Kv7.1 and reduced IKs. Our data provide a novel mechanistic insight into how the perturbed structure-function relationship of CaM variants contributes to the LQTS phenotype.