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.

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 deep learning algorithm to translate and classify cardiac electrophysiology.

The development of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) has been a critical in vitro advance in the study of patient-specific physiology, pathophysiology, and pharmacology. We designed a new deep learning multitask network approach intended to address the low throughput, high variability, and immature phenotype of the iPSC-CM platform. The rationale for combining translation and classification tasks is because the most likely application of the deep learning technology we describe here is to translate iPSC-CMs following application of a perturbation. The deep learning network was trained using simulated action potential (AP) data and applied to classify cells into the drug-free and drugged categories and to predict the impact of electrophysiological perturbation across the continuum of aging from the immature iPSC-CMs to the adult ventricular myocytes. The phase of the AP extremely sensitive to perturbation due to a steep rise of the membrane resistance was found to contain the key information required for successful network multitasking. We also demonstrated successful translation of both experimental and simulated iPSC-CM AP data validating our network by prediction of experimental drug-induced effects on adult cardiomyocyte APs by the latter.

Computational prediction of drug response in short QT syndrome type 1 based on measurements of compound effect in stem cell-derived cardiomyocytes.

Short QT (SQT) syndrome is a genetic cardiac disorder characterized by an abbreviated QT interval of the patient's electrocardiogram. The syndrome is associated with increased risk of arrhythmia and sudden cardiac death and can arise from a number of ion channel mutations. Cardiomyocytes derived from induced pluripotent stem cells generated from SQT patients (SQT hiPSC-CMs) provide promising platforms for testing pharmacological treatments directly in human cardiac cells exhibiting mutations specific for the syndrome. However, a difficulty is posed by the relative immaturity of hiPSC-CMs, with the possibility that drug effects observed in SQT hiPSC-CMs could be very different from the corresponding drug effect in vivo. In this paper, we apply a multistep computational procedure for translating measured drug effects from these cells to human QT response. This process first detects drug effects on individual ion channels based on measurements of SQT hiPSC-CMs and then uses these results to estimate the drug effects on ventricular action potentials and QT intervals of adult SQT patients. We find that the procedure is able to identify IC50 values in line with measured values for the four drugs quinidine, ivabradine, ajmaline and mexiletine. In addition, the predicted effect of quinidine on the adult QT interval is in good agreement with measured effects of quinidine for adult patients. Consequently, the computational procedure appears to be a useful tool for helping predicting adult drug responses from pure in vitro measurements of patient derived cell lines.

The phosphorylation state of both hERG and KvLQT1 mediates protein-protein interactions between these complementary cardiac potassium channel alpha subunits.

KvLQT1 and hERG are the α-subunits of the voltage-gated K+ channels which carry the cardiac repolarizing currents IKs and IKr, respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs) in cardiomyocytes. As such, protein-protein interactions between hERG and KvLQT1 may be important in normal cardiac electrophysiology, as well as in arrhythmia and sudden cardiac death. Previous phenomenological observations of functional, mutual downregulation between these complementary repolarizing currents in transgenic rabbit models and human cell culture motivate our investigations into protein-protein interactions between hERG and KvLQT1. Previous data suggest that a dynamic, physical interaction between hERG and KvLQT1 modulates the respective currents. However, the mechanism by which hERG-KvLQT1 interactions are regulated is still poorly understood. Phosphorylation is proposed to play a role since modifying the phosphorylation state of each protein has been shown to alter channel kinetics, and both hERG and KvLQT1 are targets of the Ser/Thr protein kinase PKA, activated by elevated intracellular cAMP. In this work, quantitative apFRET analyses of phosphonull and phosphomimetic hERG and KvLQT1 mutants indicate that unphosphorylated hERG does not interact with KvLQT1, suggesting that hERG phosphorylation is important for wild-type proteins to interact. For proteins already potentially interacting, phosphorylation of KvLQT1 appears to be the driving factor abrogating hERG-KvLQT1 interaction. This work increases our knowledge about hERG-KvLQT1 interactions, which may contribute to the efforts to elucidate mechanisms that underlie many types of arrhythmias, and also further characterizes novel protein-protein interactions between two distinct potassium channel families.

Enterocyte-Based Bioassay via Quantitative Combination of Proinflammatory Sentinels Specific to 8-keto-trichothecenes.

Type B 8-keto-trichothecenes are muco-active mycotoxins that exist as inevitable contaminants in cereal-based foodstuffs. Gut-associated inflammation is an early frontline response during human and animal exposure to these mycotoxins. Despite various tools for chemical identification, optimized biomonitoring of sentinel response-associated biomarkers is required to assess the specific proinflammatory actions of 8-keto-trichothecenes in the gut epithelial barrier. In the present study, intoxication with 8-keto-trichothecenes in human intestinal epithelial cells was found to trigger early response gene 1 product (EGR-1) that plays crucial roles in proinflammatory chemokine induction. In contrast, epithelial exposure to 8-keto-trichothecenes resulted in downregulated expression of nuclear factor NF-kappa-B p65 protein, a key transcription factor, during general inflammatory responses in the gut. Based on the early molecular patterns of expression, the inflammation-inducing activity of 8-keto-trichothecenes was quantified using intestinal epithelial cells with dual reporters for EGR-1 and p65 proteins. EGR-1-responsive elements were linked to luciferase reporter while p65 promoter was bound to secretory alkaline phosphatase (SEAP) reporter. In response to conventional inflammagens such as endotoxins and cytokines such as TNF-α, both luciferase and SEAP activity were elevated in a dose-dependent manner. However, as expected from the mechanistic evaluation, 8-keto-trichothecene-exposed dual reporters of luciferase and SEAP displayed contrasting expression patterns. Furthermore, 8-keto-trichothecene-elevated EGR-1-responsive luciferase activity was improved by deficiency of PSMA3, an α-type subunit of the 20S proteasome core complex for ubiquitin-dependent EGR-1 degradation. This molecular event-based dual biomonitoring in epithelial cells is a promising supplementary tool for detecting typical molecular inflammatory pathways in response to 8-keto-trichothecenes in the food matrix.

Safety assessment of metarrestin in dogs: A clinical candidate targeting a subnuclear structure unique to metastatic cancer cells.

Pancreatic cancer is a leading cause of cancer-related deaths in the U.S. Ninety percent of patients with stage IV pancreatic cancer die within one year of diagnosis due to complications of metastasis. A metastatic potential of cancer cells has been shown to be closely associated with formation of perinucleolar compartment (PNC). Metarrestin, a first-in-class PNC inhibitor, was evaluated for its toxicity, toxicokinetics, and safety pharmacology in beagle dogs following every other day oral (capsule) administration for 28 days to support its introduction into clinical trials. The study consisted of four dose groups: vehicle; 0.25, 0.75 and 1.50 mg/kg/dose. Metarrestin reached its maximum concentration in blood at 3 h (overall median Tmax) across all doses with a mean t1/2 over 168 h of 55.5 h. Dose dependent increase in systemic exposure (Cmax and AUClast) with no sex difference was observed on days 1 and 27. Metarrestin accumulated from Day 1 to Day 27 at all dose levels and in both sexes by an overall factor of about 2.34. No mortality occurred during the dosing period; however, treatment-related clinical signs of toxicity consisting of hypoactivity, shaking/shivering, thinness, irritability, salivation, abnormal gait, tremors, ataxia and intermittent seizure-like activity were seen in both sexes at mid and high dose groups. Treatment-related effects on body weight and food consumption were seen at the mid and high dose levels. Safety pharmacology study showed no treatment-related effects on blood pressure, heart rate, corrected QT, PR, RR, or QRS intervals, or respiratory function parameters (respiratory rate, tidal volume, minute volume). There were no histopathological changes observed, with the exception of transient thymic atrophy which was considered to be non-adverse. Based primarily on clinical signs of toxicity, the No Observed Adverse Effect Level (NOAEL) in dogs was considered to be 0.25 mg/kg metarrestin after every other day dosing for 28 days with a mean of male and female Cmax = 82.5 ng/mL and AUClast = 2521 h*ng/mL, on Day 27.

The influence of hERG1a and hERG1b isoforms on drug safety screening in iPSC-CMs.

The human Ether-à-go-go Related Gene (hERG) encodes the pore forming subunit of the channel that conducts the rapid delayed rectifier potassium current IKr. IKr drives repolarization in the heart and when IKr is dysfunctional, cardiac repolarization delays, the QT interval on the electrocardiogram (ECG) prolongs and the risk of developing lethal arrhythmias such as Torsade de Pointes (TdP) increases. TdP risk is incorporated in drug safety screening for cardiotoxicity where hERG is the main target since the IKr channels appear highly sensitive to blockage. hERG block is also included as an important read-out in the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative which aims to combine in vitro and in silico experiments on induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to screen for cardiotoxicity. However, the hERG channel has some unique features to consider for drug safety screening, which we will discuss in this study. The hERG channel consists of different isoforms, hERG1a and hERG1b, which individually influence the kinetics of the channel and the drug response in the human heart and in iPSC-CMs. hERG1b is often underappreciated in iPSC-CM studies, drug screening assays and in silico models, and the fact that its contribution might substantially differ between iPSC-CM and healthy but also diseased human heart, adds to this problem. In this study we show that the activation kinetics in iPSC-CMs resemble hERG1b kinetics using Cs+ as a charge carrier. Not including hERG1b in drug safety testing might underestimate the actual role of hERG1b in repolarization and drug response, and might lead to inappropriate conclusions. We stress to focus more on including hERG1b in drug safety testing concerning IKr.

In vitro safety pharmacology evaluation of 2-hydroxybenzylamine acetate.

2-hydroxybenzylamine (2-HOBA), a compound found in buckwheat, is a potent scavenger of reactive γ-ketoaldehydes, which are increased in diseases associated with inflammation and oxidative stress. While the potential of 2-HOBA is promising, studies were needed to characterize the safety of the compound before clinical trials. In a series of experiments, the risks of 2-HOBA-mediated mutagenicity and cardio-toxicity were assessed in vitro. The effects of 2-HOBA on the mRNA expression of select cytochrome P450 (CYP) enzymes were also assessed in cryopreserved human hepatocytes. Further, the distribution and metabolism of 2-HOBA in blood were determined. Our results indicate that 2-HOBA is not cytotoxic or mutagenic in vitro and does not induce the expression of CYP1A2, CYP2B6, or CYP3A4 in human hepatocytes. The results of the hERG testing showed a low risk of cardiac QT wave prolongation. Plasma protein binding and red blood cell distribution characteristics indicate low protein binding and no preferential distribution into erythrocytes. The major metabolites identified were salicylic acid and the glycoside conjugate of 2-HOBA. Together, these findings support development of 2-HOBA as a nutritional supplement and provide important information for the design of further preclinical safety studies in animals as well as for human clinical trials with 2-HOBA.

Upregulation of functional Kv11.1a isoform expression by modified U1 small nuclear RNA.

The KCNH2 or human ether-a go-go-related gene (hERG) encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier potassium current in the heart. The expression of Kv11.1 C-terminal isoforms is directed by the alternative splicing and polyadenylation of intron 9. Splicing of intron 9 leads to the formation of a functional, full-length Kv11.1a isoform and polyadenylation of intron 9 results in the production of a non-functional, C-terminally truncated Kv11.1a-USO isoform. The relative expression of Kv11.1a and Kv11.1a-USO plays an important role in regulating Kv11.1 channel function. In the heart, only one-third of KCNH2 pre-mRNA is processed to Kv11.1a due to the weak 5' splice site of intron 9. We previously showed that the weak 5' splice site is caused by sequence deviation from the consensus, and that mutations toward the consensus sequence increased the efficiency of intron 9 splicing. It is well established that 5' splice sites are recognized by complementary base-paring with U1 small nuclear RNA (U1 snRNA). In this study, we modified the sequence of U1 snRNA to increase its complementarity to the 5' splice site of KCNH2 intron 9 and observed a significant increase in the efficiency of intron 9 splicing. RNase protection assay and western blot analysis showed that modified U1 snRNA increased the expression of the functional Kv11.1a isoform and concomitantly decreased the expression of the non-functional Kv11.1a-USO isoform. In patch-clamp experiments, modified U1 snRNA significantly increased Kv11.1 current. Our findings suggest that relative expression of Kv11.1 C-terminal isoforms can be regulated by modified U1 snRNA.

4-Aminoquinoline Antimalarials Containing a Benzylmethylpyridylmethylamine Group Are Active against Drug Resistant Plasmodium falciparum and Exhibit Oral Activity in Mice.

Emergence of drug resistant Plasmodium falciparum including artemisinin-tolerant parasites highlights the need for new antimalarials. We have previously shown that dibemequines, 4-amino-7-chloroquinolines with dibenzylmethylamine (dibemethin) side chains, are efficacious. In this study, analogues in which the terminal phenyl group of the dibemethin was replaced with a 2-pyridyl group and in which the 4-amino-7-chloroquinoline was either maintained or replaced with a 4-aminoquinoline-7-carbonitrile were synthesized in an effort to improve druglikeness. These compounds exhibited significantly improved solubility and decreased lipophilicity and were potent against chloroquine-sensitive (NF54) and -resistant (Dd2 and 7G8) P. falciparum strains with 5/6 having IC50 < 100 nM against the NF54 strain. All inhibited both ß-hematin (synthetic hemozoin) formation and hemozoin formation in the parasite. Parasitemia was reduced by over 90% in P. berghei infected mice in 3/6 derivatives following oral dosing at 4 × 30 mg/kg, with microsomal metabolic stability data suggesting that this could be attributed to highly active metabolites.

Discovery of a new mexiletine-derived agonist of the hERG K+ channel.

The human Ether-a-go-go Related Gene (hERG) potassium channel plays a central role in the rapid component (IKr) of cardiac action potential repolarization phase. A large number of structurally different compounds block hERG and cause a high risk of arrhythmias. Among the drugs that block hERG channel, a few compounds have been identified as hERG channel activators. Such compounds may be useful, at least in theory, for the treatment of long term QT syndrome. Here we describe a new activator of hERG channel, named MC450. This compound is a symmetric urea, derived from (R)-mexiletine. Using patch-clamp recordings, we found that MC450 increased the activation current of hERG channel, with an EC50 of 41±4µM. Moreover MC450 caused a depolarizing shift in the voltage dependence of inactivation from -64.1±1.2mV (control), to -35.9±1.4mV, whereas it had no effect on the voltage dependence of activation. Furthermore, MC450 slowed current inactivation and the effect of MC450 was attenuated by the inactivation-impaired double mutant G628C/S631C.

Express with caution: Epitope tags and cDNA variants effects on hERG channel trafficking, half-life and function.

INTRODUCTION: Genetic mutations in KCNH2, which encodes hERG, the alpha subunit of the potassium channel responsible for the IKr current, cause long QT syndrome (LQTS), an inherited cardiac arrhythmia disorder. Electrophysiology techniques are used to correlate genotype with molecular phenotype to determine which mutations identified in patients diagnosed with LQTS are disease causing, and which are benign. These investigations are usually done using heterologous expression in cell lines, and often, epitope fusion tags are used to enable isolation and identification of the protein of interest. METHODS AND RESULTS: Here, we demonstrate through electrophysiology techniques and immunohistochemistry, that both N-terminal and C-terminal myc fusion tags may perturb hERG protein channel expression and kinetics of the IKr current. We also characterize the impact of 2 previously reported inadvertent cDNA variants on hERG channel expression and half-life. CONCLUSION: Our results underscore the importance of careful characterization of the impact of epitope fusion tags and of confirming complete sequence accuracy prior to genotype-phenotype studies for ion channel proteins such as hERG.

Cellular mechanisms for trazodone-induced cardiotoxicity.

The second-generation selective 5-HT2 receptor antagonists and reuptake inhibitors (SARIs) class antidepressants are known to have fewer cardiovascular side effects than the older ones. However, several case reports showed that trazodone, one of the second-generation SARIs, induces QT prolongation, cardiac arrhythmia, and ventricular tachycardia. Although these clinical cases suggested trazodone-induced cardiotoxicity, the toxicological actions of trazodone on cardiac action potentials (APs) beyond the human ether-a-go-go related gene (hERG) remain unclear. To elucidate the cellular mechanism for the adverse cardiac effects of trazodone, we investigated its effects on cardiac APs and ion channels using whole-cell patch clamp techniques in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and transiently transfected human embryonic kidney cells (HEK293) with cardiac ion channel complementary DNA. Trazodone dose-dependently decreased the maximum upstroke velocity (Vmax) and prolonged the AP duration, inducing early after depolarizations at 3 and 10 µM that triggered ventricular arrhythmias in hiPSC-CMs. Trazodone also inhibited all of the major ion channels (IKr, IKs, INa, and ICa), with an especially high inhibitory potency on hERG. These data indicate that the prolonged AP duration and decreased Vmax due to trazodone are mainly the result of hERG and sodium ion inhibition, and its inhibitory effects on cardiac ion channels can be exhibited in hiPSC-CMs.