Novel K+ Channel Targets in Atrial Fibrillation Drug Development--Where Are We?

There is a clear unmet medical need for new pharmacologic therapies with improved efficacy and safety for the treatment of atrial fibrillation. Considerable research efforts have been undertaken to discover and develop new safe and effective antiarrhythmic drugs that specifically target atrial K(+) channels. To realize the full value of these novel atrial-specific therapeutic drug targets, demonstration of clinical efficacy and safety is required for a new breed of atrial-selective antiarrhythmic drugs. The reward for demonstrating this in a pivotal phase III trial, on regulatory approval, will be "first-in-class" status. This article reviews the development status of new and novel K channel inhibitors currently in drug development as atrial-selective antiarrhythmics for the treatment of atrial fibrillation.

SAP97 and dystrophin macromolecular complexes determine two pools of cardiac sodium channels Nav1.5 in cardiomyocytes.

RATIONALE: The cardiac sodium channel Na(v)1.5 plays a key role in excitability and conduction. The 3 last residues of Na(v)1.5 (Ser-Ile-Val) constitute a PDZ-domain binding motif that interacts with the syntrophin-dystrophin complex. As dystrophin is absent at the intercalated discs, Na(v)1.5 could potentially interact with other, yet unknown, proteins at this site. OBJECTIVE: The aim of this study was to determine whether Na(v)1.5 is part of distinct regulatory complexes at lateral membranes and intercalated discs. METHODS AND RESULTS: Immunostaining experiments demonstrated that Na(v)1.5 localizes at lateral membranes of cardiomyocytes with dystrophin and syntrophin. Optical measurements on isolated dystrophin-deficient mdx hearts revealed significantly reduced conduction velocity, accompanied by strong reduction of Na(v)1.5 at lateral membranes of mdx cardiomyocytes. Pull-down experiments revealed that the MAGUK protein SAP97 also interacts with the SIV motif of Na(v)1.5, an interaction specific for SAP97 as no pull-down could be detected with other cardiac MAGUK proteins (PSD95 or ZO-1). Furthermore, immunostainings showed that Na(v)1.5 and SAP97 are both localized at intercalated discs. Silencing of SAP97 expression in HEK293 and rat cardiomyocytes resulted in reduced sodium current (I(Na)) measured by patch-clamp. The I(Na) generated by Na(v)1.5 channels lacking the SIV motif was also reduced. Finally, surface expression of Na(v)1.5 was decreased in silenced cells, as well as in cells transfected with SIV-truncated channels. CONCLUSIONS: These data support a model with at least 2 coexisting pools of Na(v)1.5 channels in cardiomyocytes: one targeted at lateral membranes by the syntrophin-dystrophin complex, and one at intercalated discs by SAP97.

Cholesterol modulates the recruitment of Kv1.5 channels from Rab11-associated recycling endosome in native atrial myocytes.

Cholesterol is an important determinant of cardiac electrical properties. However, underlying mechanisms are still poorly understood. Here, we examine the hypothesis that cholesterol modulates the turnover of voltage-gated potassium channels based on previous observations showing that depletion of membrane cholesterol increases the atrial repolarizing current I(Kur). Whole-cell currents and single-channel activity were recorded in rat adult atrial myocytes (AAM) or after transduction with hKv1.5-EGFP. Channel mobility and expression were studied using fluorescence recovery after photobleaching (FRAP) and 3-dimensional microscopy. In both native and transduced-AAMs, the cholesterol-depleting agent MbetaCD induced a delayed ( approximately 7 min) increase in I(Kur); the cholesterol donor LDL had an opposite effect. Single-channel recordings revealed an increased number of active Kv1.5 channels upon MbetaCD application. Whole-cell recordings indicated that this increase was not dependent on new synthesis but on trafficking of existing pools of intracellular channels whose exocytosis could be blocked by both N-ethylmaleimide and nonhydrolyzable GTP analogues. Rab11 was found to coimmunoprecipitate with hKv1.5-EGFP channels and transfection with Rab11 dominant negative (DN) but not Rab4 DN prevented the MbetaCD-induced I(Kur) increase. Three-dimensional microscopy showed a decrease in colocalization of Kv1.5 and Rab11 in MbetaCD-treated AAM. These results suggest that cholesterol regulates Kv1.5 channel expression by modulating its trafficking through the Rab11-associated recycling endosome. Therefore, this compartment provides a submembrane pool of channels readily available for recruitment into the sarcolemma of myocytes. This process could be a major mechanism for the tuning of cardiac electrical properties and might contribute to the understanding of cardiac effects of lipid-lowering drugs.

Kv4 potassium channels form a tripartite complex with the anchoring protein SAP97 and CaMKII in cardiac myocytes.

Membrane-associated guanylate kinase (MAGUK) proteins are major determinants of the organization of ion channels in the plasma membrane in various cell types. Here, we investigated the interaction between the MAGUK protein SAP97 and cardiac Kv4.2/3 channels, which account for a large part of the outward potassium current, I(to), in heart. We found that the Kv4.2 and Kv4.3 channels C termini interacted with SAP97 via a SAL amino acid sequence. SAP97 and Kv4.3 channels were colocalized in the sarcolemma of cardiomyocytes. In CHO cells, SAP97 clustered Kv4.3 channels in the plasma membrane and increased the current independently of the presence of KChIP and dipeptidyl peptidase-like protein-6. Suppression of SAP97 by using short hairpin RNA inhibited I(to) in cardiac myocytes, whereas its overexpression by using an adenovirus increased I(to). Kv4.3 channels without the SAL sequence were no longer regulated by Ca2+/calmodulin kinase (CaMK)II inhibitors. In cardiac myocytes, pull-down and coimmunoprecipitation assays showed that the Kv4 channel C terminus, SAP97, and CaMKII interact together, an interaction suppressed by SAP97 silencing and enhanced by SAP97 overexpression. In HEK293 cells, SAP97 silencing reproduced the effects of CaMKII inhibition on current kinetics and suppressed Kv4/CaMKII interactions. In conclusion, SAP97 is a major partner for surface expression and CaMKII-dependent regulation of cardiac Kv4 channels.

Rheumatic heart disease screening by echocardiography: the inadequacy of World Health Organization criteria for optimizing the diagnosis of subclinical disease.

BACKGROUND: Early case detection is vital in rheumatic heart disease (RHD) in children to minimize the risk of advanced valvular heart disease by preventive measures. The currently utilized World Health Organization (WHO) criteria for echocardiographic diagnosis of subclinical RHD emphasize the presence of pathological valve regurgitation but do not include valves with morphological features of RHD without pathological regurgitation. We hypothesized that adding morphological features to diagnostic criteria might have significant consequences in terms of case detection rates. METHODS AND RESULTS: We screened 2170 randomly selected school children aged 6 to 17 years in Maputo, Mozambique, clinically and by a portable ultrasound system. Two different echocardiographic sets of criteria for RHD were assessed: "WHO" (exclusively Doppler-based) and "combined" (Doppler and morphology-based) criteria. Independent investigators reviewed all suspected RHD cases using a higher-resolution, nonportable ultrasound system. On-site echocardiography identified 18 and 124 children with suspected RHD according to WHO and combined criteria, respectively. After consensus review, 17 were finally considered to have definite RHD according to WHO criteria, and 66 had definite RHD according to combined criteria, giving prevalence rates of 7.8 (95% confidence interval, 4.6 to 12.5) and 30.4 (95% confidence interval, 23.6 to 38.5) per 1000 children, respectively (P<0.0001, exact McNemar test). CONCLUSIONS: Important consideration should be given to echocardiographic criteria for detecting subclinical RHD because the number of cases detected may differ importantly according to the diagnostic criteria utilized. Currently recommended WHO criteria risk missing up to three quarters of cases of subclinically affected and therefore potentially treatable children with RHD.

Effect of selective IK,ACh inhibition by XAF-1407 in an equine model of tachypacing-induced persistent atrial fibrillation.

BACKGROUND AND PURPOSE: Inhibition of the G-protein gated ACh-activated inward rectifier potassium current, IK,ACh may be an effective atrial selective treatment strategy for atrial fibrillation (AF). Therefore, the anti-arrhythmic and electrophysiological properties of a novel putatively potent and highly specific IK,ACh inhibitor, XAF-1407 (3-methyl-1-[5-phenyl-4-[4-(2-pyrrolidin-1-ylethoxymethyl)-1-piperidyl]thieno[2,3-d]pyrimidin-6-yl]azetidin-3-ol), were characterised for the first time in vitro and investigated in horses with persistent AF. EXPERIMENTAL APPROACH: The pharmacological ion channel profile of XAF-1407 was investigated using cell lines expressing relevant ion channels. In addition, eleven horses were implanted with implantable cardioverter defibrillators enabling atrial tachypacing into self-sustained AF. The electrophysiological effects of XAF-1407 were investigated after serial cardioversions over a period of 1 month. Cardioversion success, drug-induced changes of atrial tissue refractoriness, and ventricular electrophysiology were assessed at baseline (day 0) and days 3, 5, 11, 17, and 29 after AF induction. KEY RESULTS: XAF-1407 potently and selectively inhibited Kir 3.1/3.4 and Kir 3.4/3.4, underlying the IK,ACh current. XAF-1407 treatment in horses prolonged atrial effective refractory period as well as decreased atrial fibrillatory rate significantly (~20%) and successfully cardioverted AF, although with a decreasing efficacy over time. XAF-1407 shortened atrioventricular-nodal refractoriness, without effect on QRS duration. QTc prolongation (4%) within 15 min of drug infusion was observed, however, without any evidence of ventricular arrhythmia. CONCLUSION AND IMPLICATIONS: XAF-1407 efficiently cardioverted sustained tachypacing-induced AF of short duration in horses without notable side effects. This supports IK,ACh inhibition as a potentially safe treatment of paroxysmal AF in horses, suggesting potential clinical value for other species including humans.

The positive frequency-dependent electrophysiological effects of the IKur inhibitor XEN-D0103 are desirable for the treatment of atrial fibrillation.

BACKGROUND: Selective inhibitors of Kv1.5 channels are being developed for the treatment of atrial fibrillation (AF). OBJECTIVES: The purpose of this study was to investigate the effects of the highly selective Kv1.5 inhibitor XEN-D0103 on human atrial action potentials (APs) at high excitation rates and to assess safety. METHODS: Intracellular APs (stimulation rates 1-5 Hz) were measured in right atrial trabeculae from patients in sinus rhythm (SR), chronic AF (cAF; AF of >6 months duration), and paroxysmal AF (pAF). The safety and tolerability of XEN-D0103 were tested in a double-blind, randomized, placebo-controlled phase 1 study. RESULTS: Depending on its concentration, XEN-D0103 elevated the plateau potential. At 1 Hz, XEN-D0103 (3 µM) shortened action potential duration at 90% repolarization (APD90) and effective refractory period (ERP) in SR preparations, but prolonged these parameters in cAF preparations. In SR and pAF preparations, the shortening effects on APD90 and ERP turned into prolongation at high rates. In cAF trabeculae, XEN-D0103 prolonged APD90 and ERP at 2 and 3 Hz. At high rates, more SR and pAF preparations failed to capture excitation in the presence of the drug than in its absence. XEN-D0103 (10 µM) did not significantly affect human ventricular APs. Even with plasma concentrations reaching 7000 ng/mL, XEN-D0103 did not increase ∆∆QTcF (QT interval corrected by the Fridericia formula) in the analysis of electrocardiograms of healthy volunteers, and no subjects receiving an active treatment had a QT or QTcF interval >450 ms, or increase in QTcF from baseline >30 ms. CONCLUSION: APD prolongation and suppression of APs by XEN-D0103 at high stimulation rates in SR and pAF tissue, but not cAF, could be of therapeutic benefit for reducing AF burden. This concept needs to be confirmed in clinical trials.

Atrial-like cardiomyocytes from human pluripotent stem cells are a robust preclinical model for assessing atrial-selective pharmacology.

Drugs targeting atrial-specific ion channels, Kv1.5 or Kir3.1/3.4, are being developed as new therapeutic strategies for atrial fibrillation. However, current preclinical studies carried out in non-cardiac cell lines or animal models may not accurately represent the physiology of a human cardiomyocyte (CM). In the current study, we tested whether human embryonic stem cell (hESC)-derived atrial CMs could predict atrial selectivity of pharmacological compounds. By modulating retinoic acid signaling during hESC differentiation, we generated atrial-like (hESC-atrial) and ventricular-like (hESC-ventricular) CMs. We found the expression of atrial-specific ion channel genes, KCNA5 (encoding Kv1.5) and KCNJ3 (encoding Kir 3.1), in hESC-atrial CMs and further demonstrated that these ion channel genes are regulated by COUP-TF transcription factors. Moreover, in response to multiple ion channel blocker, vernakalant, and Kv1.5 blocker, XEN-D0101, hESC-atrial but not hESC-ventricular CMs showed action potential (AP) prolongation due to a reduction in early repolarization. In hESC-atrial CMs, XEN-R0703, a novel Kir3.1/3.4 blocker restored the AP shortening caused by CCh. Neither CCh nor XEN-R0703 had an effect on hESC-ventricular CMs. In summary, we demonstrate that hESC-atrial CMs are a robust model for pre-clinical testing to assess atrial selectivity of novel antiarrhythmic drugs.

Activation of glibenclamide-sensitive ATP-sensitive K+ channels during ß-adrenergically induced metabolic stress produces a substrate for atrial tachyarrhythmia.

BACKGROUND: Cardiac ATP-sensitive K(+) channels have been suggested to contribute to the adaptive physiological response to metabolic challenge after ß-adrenoceptor stimulation. However, an increased atrial K(+)-conductance might be expected to be proarrhythmic. We investigated the effect of ATP-sensitive K(+) channel blockade on the electrophysiological responses to ß-adrenoceptor-induced metabolic challenge in intact atria. METHODS AND RESULTS: Atrial electrograms were recorded from the left atrial epicardial surface of Langendorff-perfused rat hearts using a 5×5 electrode array. Atrial effective refractory period and conduction velocity were measured using an S(1)-S(2) protocol. The proportion of hearts in which atrial tachyarrhythmia was produced by burst-pacing was used as an index of atrial tachyarrhythmia-inducibility. Atrial nucleotide concentrations were measured by high performance liquid chromatography. Perfusion with ≥10(-9) mol/L of the ß-adrenoceptor agonist, isoproterenol (ISO), resulted in a concentration-dependent reduction of atrial effective refractory period and conduction velocity. The ISO-induced changes produced a proarrhythmic substrate such that atrial tachyarrhythmia could be induced by burst-pacing. Atrial [ATP] was significantly reduced by ISO (10(-6) mol/L). Perfusion with either of the ATP-sensitive K(+) channel blockers, glibenclamide (10(-5) mol/L) or tolbutamide (10(-3) mol/L), in the absence of ISO had no effect on basal atrial electrophysiology. On the other hand, the proarrhythmic substrate induced by 10(-6) mol/L ISO was abolished by either of the sulfonylureas, which prevented induction of atrial tachyarrhythmia. CONCLUSIONS: Atrial ATP-sensitive K(+) channels activate in response to ß-adrenergic metabolic stress in Langendorff-perfused rat hearts, resulting in a proarrhythmic substrate.

The anchoring protein SAP97 retains Kv1.5 channels in the plasma membrane of cardiac myocytes.

Membrane- associated guanylate kinase proteins (MAGUKs) are important determinants of localization and organization of ion channels into specific plasma membrane domains. However, their exact role in channel function and cardiac excitability is not known. We examined the effect of synapse-associated protein 97 (SAP97), a MAGUK abundantly expressed in the heart, on the function and localization of Kv1.5 subunits in cardiac myocytes. Recombinant SAP97 or Kv1.5 subunits tagged with green fluorescent protein (GFP) were overexpressed in rat neonatal cardiac myocytes and in Chinese hamster ovary (CHO) cells from adenoviral or plasmidic vectors. Immunocytochemistry, fluorescence recovery after photobleaching, and patch-clamp techniques were used to study the effects of SAP97 on the localization, mobility, and function of Kv1.5 subunits. Adenovirus-mediated SAP97 overexpression in cardiac myocytes resulted in the clustering of endogenous Kv1.5 subunits at myocyte-myocyte contacts and an increase in both the maintained component of the outward K(+) current, I(Kur) (5.64 +/- 0.57 pA/pF in SAP97 myocytes vs. 3.23 +/- 0.43 pA/pF in controls) and the number of 4-aminopyridine-sensitive potassium channels in cell-attached membrane patches. In live myocytes, GFP-Kv1.5 subunits were mobile and organized in clusters at the basal plasma membrane, whereas SAP97 overexpression reduced their mobility. In CHO cells, Kv1.5 channels were diffusely distributed throughout the cell body and freely mobile. When coexpressed with SAP97, Kv subunits were organized in plaquelike clusters and poorly mobile. In conclusion, SAP97 regulates the K(+) current in cardiac myocytes by retaining and immobilizing Kv1.5 subunits in the plasma membrane. This new regulatory mechanism may contribute to the targeting of Kv channels in cardiac myocytes.