LNK/SH2B3 loss of function increases susceptibility to murine and human atrial fibrillation.

AIMS: The lymphocyte adaptor protein (LNK) is a negative regulator of cytokine and growth factor signaling. The rs3184504 variant in SH2B3 reduces LNK function and is linked to cardiovascular, inflammatory, and hematologic disorders including stroke. In mice, deletion of Lnk causes inflammation and oxidative stress. We hypothesized that Lnk-/- mice are susceptible to atrial fibrillation (AF) and that rs3184504 is associated with AF and AF-related stroke in humans. During inflammation, reactive lipid dicarbonyls are a major component of oxidative injury, and we further hypothesized that these mediators are critical drivers of the AF substrate in Lnk-/- mice. METHODS AND RESULTS: Lnk-/- or wild-type (WT) mice were treated with vehicle or 2-hydroxybenzylamine (2-HOBA), a dicarbonyl scavenger, for 3 months. Compared to WT, Lnk-/- mice displayed increased AF duration that was prevented by 2-HOBA. In the Lnk-/- atria, action potentials were prolonged with reduced transient outward K+ current, increased late Na+ current, and reduced peak Na+ current, proarrhythmic effects that were inhibited by 2-HOBA. Mitochondrial dysfunction, especially for complex I, was evident in Lnk-/- atria, while scavenging lipid dicarbonyls prevented this abnormality. Tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were elevated in Lnk-/- plasma and atrial tissue, respectively, both of which caused electrical and bioenergetic remodeling in vitro. Inhibition of soluble TNF-α prevented electrical remodeling and AF susceptibility, while IL-1ß inhibition improved mitochondrial respiration but had no effect on AF susceptibility. In a large database of genotyped patients, rs3184504 was associated with AF, as well as AF-related stroke. CONCLUSIONS: These findings identify a novel role for LNK in the pathophysiology of AF in both experimental mice and in humans. Moreover, reactive lipid dicarbonyls are critical to the inflammatory AF substrate in Lnk-/- mice and mediate the proarrhythmic effects of pro-inflammatory cytokines, primarily through electrical remodeling.

ent-Verticilide B1 Inhibits Type 2 Ryanodine Receptor Channels and is Antiarrhythmic in Casq2 -/- Mice.

Intracellular Ca2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent-(+)-verticilide (ent-1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product (nat-(-)-verticilide). Here, we examined its 18-membered ring-size oligomer (ent-verticilide B1; "ent-B1") in RyR2 single channel and [3H]ryanodine binding assays, and in Casq2 -/- cardiomyocytes and mice, a gene-targeted model of SCD. ent-B1 inhibited RyR2 single channels and RyR2-mediated spontaneous Ca2+ release in Casq2 -/- cardiomyocytes with sub-micromolar potency. ent-B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent-B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 minutes and half-life of 45 minutes after intraperitoneal administration of 3 mg/kg in mice. In vivo, ent-B1 significantly reduced catecholamine-induced ventricular arrhythmias in Casq2 -/- mice in a dose-dependent manner. Hence, we have identified a novel chemical entity - ent-B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. SIGNIFICANCE STATEMENT: The cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.

Eicosanoid-Regulated Myeloid ENaC and Isolevuglandin Formation in Human Salt-Sensitive Hypertension.

BACKGROUND: The mechanisms by which salt increases blood pressure in people with salt sensitivity remain unclear. Our previous studies found that high sodium enters antigen-presenting cells (APCs) via the epithelial sodium channel and leads to the production of isolevuglandins and hypertension. In the current mechanistic clinical study, we hypothesized that epithelial sodium channel-dependent isolevuglandin-adduct formation in APCs is regulated by epoxyeicosatrienoic acids (EETs) and leads to salt-sensitive hypertension in humans. METHODS: Salt sensitivity was assessed in 19 hypertensive subjects using an inpatient salt loading and depletion protocol. Isolevuglandin-adduct accumulation in APCs was analyzed using flow cytometry. Gene expression in APCs was analyzed using cellular indexing of transcriptomes and epitopes by sequencing analysis of blood mononuclear cells. Plasma and urine EETs were measured using liquid chromatography-mass spectrometry. RESULTS: Baseline isolevuglandin+ APCs correlated with higher salt-sensitivity index. Isolevuglandin+ APCs significantly decreased from salt loading to depletion with an increasing salt-sensitivity index. We observed that human APCs express the epithelial sodium channel δ subunit, SGK1 (salt-sensing kinase serum/glucocorticoid kinase 1), and cytochrome P450 2S1. We found a direct correlation between baseline urinary 14,15 EET and salt-sensitivity index, whereas changes in urinary 14,15 EET negatively correlated with isolevuglandin+ monocytes from salt loading to depletion. Coincubation with 14,15 EET inhibited high-salt-induced increase in isolevuglandin+ APC. CONCLUSIONS: Isolevuglandin formation in APCs responds to acute changes in salt intake in salt-sensitive but not salt-resistant people with hypertension, and this may be regulated by renal 14,15 EET. Baseline levels of isolevuglandin+ APCs or urinary 14,15 EET may provide diagnostic tools for salt sensitivity without a protocol of salt loading.

RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET.

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca2+-release channels contributes to heart failure and arrhythmias. Reducing the RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer (ent) of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca2+ and has in vivo efficacy against atrial and ventricular arrhythmia. To determine the ent-verticilide structural mode of action on RyR2 and guide its further development via medicinal chemistry structure-activity relationship studies, here, we used fluorescence lifetime (FLT)-measurements of Förster resonance energy transfer (FRET) in HEK293 cells expressing human RyR2. For these studies, we used an RyR-specific FRET molecular-toolkit and computational methods for trilateration (i.e., using distances to locate a point of interest). Multiexponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptor-labeled ent-verticilide yielded distance relationships placing the acceptor probe at two candidate loci within the RyR2 cryo-EM map. One locus is within the Ry12 domain (at the corner periphery of the RyR2 tetrameric complex). The other locus is sandwiched at the interface between helical domain 1 and the SPRY3 domain. These findings document RyR2-target engagement by ent-verticilide, reveal new insight into the mechanism of action of this new class of RyR2-targeting drug candidate, and can serve as input in future computational determinations of the ent-verticilide binding site on RyR2 that will inform structure-activity studies for lead optimization.

ent -Verticilide B1 inhibits type 2 ryanodine receptor channels and is antiarrhythmic in Casq2-/- mice.

Ca 2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca 2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent- (+)-verticilide ( ent -1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product ( nat -(-)-verticilide). Here, we examined its 18-membered ring-size oligomer ( ent -verticilide B1; " ent -B1") in single RyR2 channel assays, [ 3 H]ryanodine binding assays, and in Casq2 -/- cardiomyocytes and mice, a gene-targeted model of SCD. ent -B1 inhibited RyR2 single-channels and [ 3 H]ryanodine binding with low micromolar potency, and RyR2-mediated spontaneous Ca 2+ release in Casq2-/- cardiomyocytes with sub-micromolar potency. ent -B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent -B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 min and half-life of 45 min after intraperitoneal administration of 3 mg/kg in mice. Both 3 mg/kg and 30 mg/kg ent -B1 significantly reduced catecholamine-induced ventricular arrhythmia in Casq2-/- mice. Hence, we have identified a novel chemical entity - ent -B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. Significance statement: The cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.

RyR2 inhibition with dantrolene is antiarrhythmic, prevents further pathological remodeling, and improves cardiac function in chronic ischemic heart disease.

Diastolic Ca2+ leak due to cardiac ryanodine receptor (RyR2) hyperactivity has been widely documented in chronic ischemic heart disease (CIHD) and may contribute to ventricular tachycardia (VT) risk and progressive left-ventricular (LV) remodeling. Here we test the hypothesis that targeting RyR2 hyperactivity can suppress VT inducibility and progressive heart failure in CIHD by the RyR2 inhibitor dantrolene. METHODS AND RESULTS: CIHD was induced in C57BL/6 J mice by left coronary artery ligation. Four weeks later, mice were randomized to either acute or chronic (6 weeks via implanted osmotic pump) treatment with dantrolene or vehicle. VT inducibility was assessed by programmed stimulation in vivo and in isolated hearts. Electrical substrate remodeling was assessed by optical mapping. Ca2+ sparks and spontaneous Ca2+ releases were measured in isolated cardiomyocytes. Cardiac remodeling was quantified by histology and qRT-PCR. Cardiac function and contractility were measured using echocardiography. Compared to vehicle, acute dantrolene treatment reduced VT inducibility. Optical mapping demonstrated reentrant VT prevention by dantrolene, which normalized the shortened refractory period (VERP) and prolonged action potential duration (APD), preventing APD alternans. In single CIHD cardiomyocytes, dantrolene normalized RyR2 hyperactivity and prevented spontaneous intracellular Ca2+ release. Chronic dantrolene treatment not only reduced VT inducibility but also reduced peri-infarct fibrosis and prevented further progression of LV dysfunction in CIHD mice. CONCLUSIONS: RyR2 hyperactivity plays a mechanistic role for VT risk, post-infarct remodeling, and contractile dysfunction in CIHD mice. Our data provide proof of concept for the anti-arrhythmic and anti-remodeling efficacy of dantrolene in CIHD.

High-throughput functional mapping of variants in an arrhythmia gene, KCNE1, reveals novel biology.

Background: KCNE1 encodes a 129-residue cardiac potassium channel (IKs) subunit. KCNE1 variants are associated with long QT syndrome and atrial fibrillation. However, most variants have insufficient evidence of clinical consequences and thus limited clinical utility. Results: Here, we demonstrate the power of variant effect mapping, which couples saturation mutagenesis with high-throughput sequencing, to ascertain the function of thousands of protein coding KCNE1 variants. We comprehensively assayed KCNE1 variant cell surface expression (2,554/2,709 possible single amino acid variants) and function (2,539 variants). We identified 470 loss-of-surface expression and 588 loss-of-function variants. Out of the 588 loss-of-function variants, only 155 had low cell surface expression. The latter half of the protein is dispensable for protein trafficking but essential for channel function. 22 of the 30 KCNE1 residues (73%) highly intolerant of variation were in predicted close contact with binding partners KCNQ1 or calmodulin. Our data were highly concordant with gold standard electrophysiological data (ρ = -0.65), population and patient cohorts (32/38 concordant variants), and computational metrics (ρ = -0.55). Our data provide moderate-strength evidence for the ACMG/AMP functional criteria for benign and pathogenic variants. Conclusions: Comprehensive variant effect maps of KCNE1 can both provide insight into IKs channel biology and help reclassify variants of uncertain significance.

The selective RyR2 inhibitor ent-verticilide suppresses atrial fibrillation susceptibility caused by Pitx2 deficiency.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a major cause of stroke and morbidity. The strongest genetic risk factors for AF in humans are variants on chromosome 4q25, near the paired-like homeobox transcription factor 2 gene PITX2. Although mice deficient in Pitx2 (Pitx2+/-) have increased AF susceptibility, the mechanism remains controversial. Recent evidence has implicated hyperactivation of the cardiac ryanodine receptor (RyR2) in Pitx2 deficiency, which may be associated with AF susceptibility. We investigated pacing-induced AF susceptibility and spontaneous Ca2+ release events in Pitx2 haploinsufficient (+/-) mice and isolated atrial myocytes to test the hypothesis that hyperactivity of RyR2 increases susceptibility to AF, which can be prevented by a potent and selective RyR2 channel inhibitor, ent-verticilide. Compared with littermate wild-type Pitx2+/+, the frequency of Ca2+ sparks and spontaneous Ca2+ release events increased in permeabilized and intact atrial myocytes from Pitx2+/- mice. Atrial burst pacing consistently increased the incidence and duration of AF in Pitx2+/- mice. The RyR2 inhibitor ent-verticilide significantly reduced the frequency of spontaneous Ca2+ release in intact atrial myocytes and attenuated AF susceptibility with reduced AF incidence and duration. Our data demonstrate that RyR2 hyperactivity enhances SR Ca2+ leak and AF inducibility in Pitx2+/- mice via abnormal Ca2+ handling. Therapeutic targeting of hyperactive RyR2 in AF using ent-verticilide may be a viable mechanism-based approach to treat atrial arrhythmias caused by Pitx2 deficiency.

In Vivo Pharmacokinetic and Pharmacodynamic Properties of the Antiarrhythmic Molecule ent-Verticilide.

The unnatural verticilide enantiomer (ent-verticilide) is a selective and potent inhibitor of cardiac ryanodine receptor (RyR2) calcium release channels and exhibits antiarrhythmic activity in a murine model of catecholaminergic polymorphic ventricular tachycardia (CPVT). To determine verticilide's pharmacokinetic and pharmacodynamic properties in vivo, we developed a bioassay to measure nat- and ent-verticilide in murine plasma and correlated plasma concentrations with antiarrhythmic efficacy in a mouse model of CPVT. nat-Verticilide rapidly degraded in plasma in vitro, showing >95% degradation within 5 minutes, whereas ent-verticilide showed <1% degradation over 6 hours. Plasma was collected from mice following intraperitoneal administration of ent-verticilide at two doses (3 mg/kg, 30 mg/kg). Peak C max and area under the plasma-concentration time curve (AUC) scaled proportionally to dose, and the half-life was 6.9 hours for the 3-mg/kg dose and 6.4 hours for the 30-mg/kg dose. Antiarrhythmic efficacy was examined using a catecholamine challenge protocol at time points ranging from 5 to 1440 minutes after intraperitoneal dosing. ent-Verticilide inhibited ventricular arrhythmias as early as 7 minutes after administration in a concentration-dependent manner, with an estimated potency (IC50) of 266 ng/ml (312 nM) and an estimated maximum inhibitory effect of 93.5%. Unlike the US Food and Drug Administration-approved pan-RyR blocker dantrolene, the RyR2-selective blocker ent-verticilide (30 mg/kg) did not reduce skeletal muscle strength in vivo. We conclude that ent-verticilide has favorable pharmacokinetic properties and reduces ventricular arrhythmias with an estimated potency in the nanomolar range, warranting further drug development. SIGNIFICANCE STATEMENT: ent-Verticilide has therapeutic potential to treat cardiac arrhythmias, but little is known about its pharmacological profile in vivo. The primary purpose of this study is to determine the systemic exposure and pharmacokinetics of ent-verticilide in mice and estimate its efficacy and potency in vivo. The current work suggests ent-verticilide has favorable pharmacokinetic properties and reduces ventricular arrhythmias with an estimated potency in the nanomolar range, warranting further drug development.

Structure-Activity Relationships for the N-Me- Versus N-H-Amide Modification to Macrocyclic ent-Verticilide Antiarrhythmics.

The synthesis of all N-Me and N-H analogues of ent-verticilide is described, enabling a structure-activity relationship study based on cardiac ryanodine receptor (RyR2) calcium ion channel inhibition. The use of permeabilized cardiomyocytes allowed us to correlate the degree of N-methylation with activity without concern for changes in passive membrane permeability that these modifications can cause. A key hypothesis was that the minimal pharmacophore may be repeated in this cyclic oligomeric octadepsipeptide (a 24-membered macrocycle), opening the possibility that target engagement will not necessarily be lost with a single N-Me → N-H modification. The effect in the corresponding 18-membered ring oligomer (ent-verticilide B1) was also investigated. We report here that a high degree of N-methyl amide content is critical for activity in the ent-verticilide series but not entirely so for the ent-verticilide B1 series.

Impaired Dynamic Sarcoplasmic Reticulum Ca Buffering in Autosomal Dominant CPVT2.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal cardiac arrhythmia syndrome triggered by catecholamines released during exercise, stress, or sudden emotion. Variants in the calsequestrin-2 gene (CASQ2), encoding the major calcium (Ca) binding protein in the sarcoplasmic reticulum (SR), are the second most common cause of CPVT. Recently, several CASQ2 gene variants, such as CASQ2-K180R, have been linked to an autosomal dominant form of Casq2-linked CPVT (CPVT2), but the underlying mechanism is not known. METHODS: A K180R mouse model was generated using CRIPSR/Cas9. Heterozygous and homozygous K180R mice were studied using telemetry ECG recordings in vivo. Ventricular cardiomyocytes were isolated and studied using fluorescent Ca indicators and patch clamp. Expression levels and localization of SR Ca-handling proteins were evaluated using Western blotting and immunostaining. Intra-SR Ca kinetics were quantified using low-affinity Ca indicators. RESULTS: K180R mice exhibit an autosomal dominant CPVT phenotype following exercise or catecholamine stress. Upon catecholamine stress, K180R ventricular cardiomyocytes exhibit increased spontaneous SR Ca release events, triggering delayed afterdepolarizations and spontaneous beats. K180R had no effect on levels of Casq2, Casq2 polymers, or other SR Ca-handling proteins. Intra-SR Ca measurements revealed that K180R impaired dynamic intra-SR Ca buffering, resulting in a more rapid rise of free Ca in the SR during diastole. Steady-state SR Ca buffering and total SR Ca content were not changed. Consistent with the reduced dynamic intra-SR buffering, K180R causes reduced SR Ca release refractoriness. CONCLUSIONS: CASQ2-K180R causes CPVT2 via a heretofore unknown mechanism that differs from CASQ2 variants associated with autosomal recessive CPVT2. Unlike autosomal recessive CASQ2 variants, K180R impairs the dynamic buffering of Ca within the SR without affecting total SR Ca content or Casq2 protein levels. Our data provide insight into the molecular mechanism underlying autosomal dominant CPVT2.

Animal Models to Study Cardiac Arrhythmias.

Cardiac arrhythmias are a significant cause of morbidity and mortality worldwide, accounting for 10% to 15% of all deaths. Although most arrhythmias are due to acquired heart disease, inherited channelopathies and cardiomyopathies disproportionately affect children and young adults. Arrhythmogenesis is complex, involving anatomic structure, ion channels and regulatory proteins, and the interplay between cells in the conduction system, cardiomyocytes, fibroblasts, and the immune system. Animal models of arrhythmia are powerful tools for studying not only molecular and cellular mechanism of arrhythmogenesis but also more complex mechanisms at the whole heart level, and for testing therapeutic interventions. This review summarizes basic and clinical arrhythmia mechanisms followed by an in-depth review of published animal models of genetic and acquired arrhythmia disorders.

Exercise Causes Arrhythmogenic Remodeling of Intracellular Calcium Dynamics in Plakophilin-2-Deficient Hearts.

BACKGROUND: Exercise training, and catecholaminergic stimulation, increase the incidence of arrhythmic events in patients affected with arrhythmogenic right ventricular cardiomyopathy correlated with plakophilin-2 (PKP2) mutations. Separate data show that reduced abundance of PKP2 leads to dysregulation of intracellular Ca2+ (Ca2+i) homeostasis. Here, we study the relation between excercise, catecholaminergic stimulation, Ca2+i homeostasis, and arrhythmogenesis in PKP2-deficient murine hearts. METHODS: Experiments were performed in myocytes from a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout murine line (PKP2cKO). For training, mice underwent 75 minutes of treadmill running once per day, 5 days each week for 6 weeks. We used multiple approaches including imaging, high-resolution mass spectrometry, electrocardiography, and pharmacological challenges to study the functional properties of cells/hearts in vitro and in vivo. RESULTS: In myocytes from PKP2cKO animals, training increased sarcoplasmic reticulum Ca2+ load, increased the frequency and amplitude of spontaneous ryanodine receptor (ryanodine receptor 2)-mediated Ca2+ release events (sparks), and changed the time course of sarcomeric shortening. Phosphoproteomics analysis revealed that training led to hyperphosphorylation of phospholamban in residues 16 and 17, suggesting a catecholaminergic component. Isoproterenol-induced increase in Ca2+i transient amplitude showed a differential response to ß-adrenergic blockade that depended on the purported ability of the blockers to reach intracellular receptors. Additional experiments showed significant reduction of isoproterenol-induced Ca2+i sparks and ventricular arrhythmias in PKP2cKO hearts exposed to an experimental blocker of ryanodine receptor 2 channels. CONCLUSIONS: Exercise disproportionately affects Ca2+i homeostasis in PKP2-deficient hearts in a manner facilitated by stimulation of intracellular ß-adrenergic receptors and hyperphosphorylation of phospholamban. These cellular changes create a proarrhythmogenic state that can be mitigated by ryanodine receptor 2 blockade. Our data unveil an arrhythmogenic mechanism for exercise-induced or catecholaminergic life-threatening arrhythmias in the setting of PKP2 deficit. We suggest that membrane-permeable ß-blockers are potentially more efficient for patients with arrhythmogenic right ventricular cardiomyopathy, highlight the potential for ryanodine receptor 2 channel blockers as treatment for the control of heart rhythm in the population at risk, and propose that PKP2-dependent and phospholamban-dependent arrhythmogenic right ventricular cardiomyopathy-related arrhythmias have a common mechanism.

A High-Throughput Screening Assay to Identify Drugs that Can Treat Long QT Syndrome Caused by Trafficking-Deficient KV11.1 (hERG) Variants.

Loss-of-function (LOF) variants in the KV11.1 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 KV11.1 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 KV11.1 variant in 384-well plates. Assay quality was assessed using Z prime (Z') scores comparing vehicle to E-4031, a drug that increases KV11.1 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 KV11.1-G601S (KV11.1-G601S-G965*X) with the addition of 2) KV11.1 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 KV11.1 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 KV11.1 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 KV11.1 variants. SIGNIFICANCE STATEMENT: This manuscript reports the development of a high-throughput assay (thallium flux) to identify drugs that can increase function in KV11.1 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.

The Purkinje-myocardial junction is the anatomic origin of ventricular arrhythmia in CPVT.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome caused by gene mutations that render RYR2 Ca release channels hyperactive, provoking spontaneous Ca release and delayed afterdepolarizations (DADs). What remains unknown is the cellular source of ventricular arrhythmia triggered by DADs: Purkinje cells in the conduction system or ventricular cardiomyocytes in the working myocardium. To answer this question, we used a genetic approach in mice to knock out cardiac calsequestrin either in Purkinje cells or in ventricular cardiomyocytes. Total loss of calsequestrin in the heart causes a severe CPVT phenotype in mice and humans. We found that loss of calsequestrin only in ventricular myocytes produced a full-blown CPVT phenotype, whereas mice with loss of calsequestrin only in Purkinje cells were comparable to WT mice. Subendocardial chemical ablation or restoration of calsequestrin expression in subendocardial cardiomyocytes neighboring Purkinje cells was sufficient to protect against catecholamine-induced arrhythmias. In silico modeling demonstrated that DADs in ventricular myocardium can trigger full action potentials in the Purkinje fiber, but not vice versa. Hence, ectopic beats in CPVT are likely generated at the Purkinje-myocardial junction via a heretofore unrecognized tissue mechanism, whereby DADs in the ventricular myocardium trigger full action potentials in adjacent Purkinje cells.

Ring Size as an Independent Variable in Cyclooligomeric Depsipeptide Antiarrhythmic Activity.

Hit-to-lead studies employ a variety of strategies to optimize binding to a target of interest. When a structure for the target is available, hypothesis-driven structure-activity relationships (SAR) are a powerful strategy for refining the pharmacophore to achieve robust binding and selectivity characteristics necessary to identify a lead compound. Recrafting the three-dimensional space occupied by a small molecule, optimization of hydrogen bond contacts, and enhancing local attractive interactions are traditional approaches in medicinal chemistry. Ring size, however, is rarely able to be leveraged as an independent variable because most hits lack the symmetry required for such a study. Our discovery that the cyclic oligomeric depsipeptide ent-verticilide inhibits mammalian cardiac ryanodine receptor calcium release channels with submicromolar potency provided an opportunity to explore ring size as a variable, independent of other structural or functional group changes. We report here that ring size can be a critical independent variable, suggesting that modest conformational changes alone can dramatically affect potency.

Assessment of dynamic cardiac repolarization and contractility in patients with hypertrophic cardiomyopathy.

AIMS: Arrhythmia mechanisms in hypertrophic cardiomyopathy remain uncertain. Preclinical models suggest hypertrophic cardiomyopathy-linked mutations perturb sarcomere length-dependent activation, alter cardiac repolarization in rate-dependent fashion and potentiate triggered electrical activity. This study was designed to assess rate-dependence of clinical surrogates of contractility and repolarization in humans with hypertrophic cardiomyopathy. METHODS: All participants had a cardiac implantable device capable of atrial pacing. Cases had clinical diagnosis of hypertrophic cardiomyopathy, controls were age-matched. Continuous electrocardiogram and blood pressure were recorded during and immediately after 30 second pacing trains delivered at increasing rates. RESULTS: Nine hypertrophic cardiomyopathy patients and 10 controls were enrolled (47% female, median 55 years), with similar baseline QRS duration, QT interval and blood pressure. Median septal thickness in hypertrophic cardiomyopathy patients was 18mm; 33% of hypertrophic cardiomyopathy patients had peak sub-aortic velocity >50mmHg. Ventricular ectopy occurred during or immediately after pacing trains in 4/9 hypertrophic cardiomyopathy patients and 0/10 controls (P = 0.03). During delivery of steady rate pacing across a range of cycle lengths, the QT-RR relationship was not statistically different between HCM and control groups; no differences were seen in subgroup analysis of patients with or without intact AV node conduction. Similarly, there was no difference between groups in the QT interval of the first post-pause recovery beat after pacing trains. No statistically significant differences were seen in surrogate measures for cardiac contractility. CONCLUSION: Rapid pacing trains triggered ventricular ectopy in hypertrophic cardiomyopathy patients, but not controls. This finding aligns with pre-clinical descriptions of excessive cardiomyocyte calcium loading during rapid pacing, increased post-pause sarcoplasmic reticulum calcium release, and subsequent calcium-triggered activity. Normal contractility at all diastolic intervals argues against clinical significance of altered length-dependent myofilament activation.

RYR2 Channel Inhibition Is the Principal Mechanism of Flecainide Action in CPVT.

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.

Intrinsically disordered HAX-1 regulates Ca2+ cycling by interacting with lipid membranes and the phospholamban cytoplasmic region.

Hematopoietic-substrate-1 associated protein X-1 (HAX-1) is a 279 amino acid protein expressed ubiquitously. In cardiac muscle, HAX-1 was found to modulate the sarcoendoplasmic reticulum calcium ATPase (SERCA) by shifting its apparent Ca2+ affinity (pCa). It has been hypothesized that HAX-1 binds phospholamban (PLN), enhancing its inhibitory function on SERCA. HAX-1 effects are reversed by cAMP-dependent protein kinase A that phosphorylates PLN at Ser16. To date, the molecular mechanisms for HAX-1 regulation of the SERCA/PLN complex are still unknown. Using enzymatic, in cell assays, circular dichroism, and NMR spectroscopy, we found that in the absence of a binding partner HAX-1 is essentially disordered and adopts a partial secondary structure upon interaction with lipid membranes. Also, HAX-1 interacts with the cytoplasmic region of monomeric and pentameric PLN as detected by NMR and in cell FRET assays, respectively. We propose that the regulation of the SERCA/PLN complex by HAX-1 is mediated by its interactions with lipid membranes, adding another layer of control in Ca2+ homeostatic balance in the heart muscle.