Ultra-high throughput-based screening for the discovery of antiplatelet drugs affecting receptor dependent calcium signaling dynamics.

Distinct platelet activation patterns are elicited by the tyrosine kinase-linked collagen receptor glycoprotein VI (GPVI) and the G-protein coupled protease-activated receptors (PAR1/4) for thrombin. This is reflected in the different platelet Ca2+ responses induced by the GPVI agonist collagen-related peptide (CRP) and the PAR1/4 agonist thrombin. Using a 96 well-plate assay with human Calcium-6-loaded platelets and a panel of 22 pharmacological inhibitors, we assessed the cytosolic Ca2+ signaling domains of these receptors and developed an automated Ca2+ curve algorithm. The algorithm was used to evaluate an ultra-high throughput (UHT) based screening of 16,635 chemically diverse small molecules with orally active physicochemical properties for effects on platelets stimulated with CRP or thrombin. Stringent agonist-specific selection criteria resulted in the identification of 151 drug-like molecules, of which three hit compounds were further characterized. The dibenzyl formamide derivative ANO61 selectively modulated thrombin-induced Ca2+ responses, whereas the aromatic sulfonyl imidazole AF299 and the phenothiazine ethopropazine affected CRP-induced responses. Platelet functional assays confirmed selectivity of these hits. Ethopropazine retained its inhibitory potential in the presence of plasma, and suppressed collagen-dependent thrombus buildup at arterial shear rate. In conclusion, targeting of platelet Ca2+ signaling dynamics in a screening campaign has the potential of identifying novel platelet-inhibiting molecules.

The Purkinje network plays a major role in low-energy ventricular defibrillation.

BACKGROUND: During ventricular fibrillation (VF), targeting the excitable gap (EG) of reentry throughout the myocardium with low-energy surface stimulation shows promise for painless defibrillation. However, the Purkinje network may provide alternative pathways for reentry to evade termination. This study investigates the role of the Purkinje network in painless defibrillation. METHODS: In a computational human biventricular model featuring a Purkinje network, VF was initiated with 4 Hz epicardial pacing. Defibrillation was attempted by stimulating myocardial surface EG with a low-energy 2 ms duration pulse at 2x stimulus capture, which was administered at coupling intervals incremented by 0.25 s between 0.25 and 5 s after VF initiation. Defibrillation was accomplished if reentry ceased ≤ 1 s after the defibrillation pulse. The protocol was repeated with the Purkinje network and myocardial surface EG stimulated simultaneously, and again after uncoupling the Purkinje network from the myocardium. RESULTS: VF with the Purkinje network coupled and uncoupled had comparable dominant frequency in the left (3.81 ± 0.44 versus 3.77 ± 0.53 Hz) and right (3.80 ± 0.37 versus 3.76 ± 0.48 Hz) ventricles. When uncoupling the Purkinje network, myocardial surface EG stimulation terminated VF for all defibrillation pulses. When coupled, myocardial EG surface stimulation terminated VF for only 55% of the defibrillation pulses, but improved to 100% when stimulated simultaneously with Purkinje network EG. Defibrillation failures were attributed to EG evading stimulation in the Purkinje network. CONCLUSIONS: Defibrillation that exclusively targets myocardium can fail due to accessory pathways in the Purkinje network that allow for reentrant activity to evade termination and maintain VF. Painless defibrillation strategies should be adapted to include the Purkinje network.

Effective termination of atrial fibrillation by SK channel inhibition is associated with a sudden organization of fibrillatory conduction.

AIMS: Pharmacological termination of atrial fibrillation (AF) remains a challenge due to limited efficacy and potential ventricular proarrhythmic effects of antiarrhythmic drugs. SK channels are proposed as atrial-specific targets in the treatment of AF. Here, we investigated the effects of the new SK channel inhibitor AP14145. METHODS AND RESULTS: Eight goats were implanted with pericardial electrodes for induction of AF (30 days). In an open-chest study, the atrial conduction velocity (CV) and effective refractory period (ERP) were measured during pacing. High-density mapping of both atrial free-walls was performed during AF and conduction properties were assessed. All measurements were performed at baseline and during AP14145 infusion [10 mg/kg/h (n = 1) or 20 mg/kg/h (n = 6)]. At an infusion rate of 20 mg/kg/h, AF terminated in five of six goats. AP14145 profoundly increased ERP and reduced CV during pacing. AP14145 increased spatiotemporal instability of conduction at short pacing cycle lengths. Atrial fibrillation cycle length and pathlength (AF cycle length × CV) underwent a strong dose-dependent prolongation. Conduction velocity during AF remained unchanged and conduction patterns remained complex until the last seconds before AF termination, during which a sudden and profound organization of fibrillatory conduction occurred. CONCLUSION: AP14145 provided a successful therapy for termination of persistent AF in goats. During AF, AP14145 caused an ERP and AF cycle length prolongation. AP14145 slowed CV during fast pacing but did not lead to a further decrease during AF. Termination of AF was preceded by an abrupt organization of AF with a decline in the number of fibrillation waves.

Synergistic antiarrhythmic effect of inward rectifier current inhibition and pulmonary vein isolation in a 3D computer model for atrial fibrillation.

AIMS: Recent clinical studies showed that antiarrhythmic drug (AAD) treatment and pulmonary vein isolation (PVI) synergistically reduce atrial fibrillation (AF) recurrences after initially successful ablation. Among newly developed atrial-selective AADs, inhibitors of the G-protein-gated acetylcholine-activated inward rectifier current (IKACh) were shown to effectively suppress AF in an experimental model but have not yet been evaluated clinically. We tested in silico whether inhibition of inward rectifier current or its combination with PVI reduces AF inducibility. METHODS AND RESULTS: We simulated the effect of inward rectifier current blockade (IK blockade), PVI, and their combination on AF inducibility in a detailed three-dimensional model of the human atria with different degrees of fibrosis. IK blockade was simulated with a 30% reduction of its conductivity. Atrial fibrillation was initiated using incremental pacing applied at 20 different locations, in both atria. IK blockade effectively prevented AF induction in simulations without fibrosis as did PVI in simulations without fibrosis and with moderate fibrosis. Both interventions lost their efficacy in severe fibrosis. The combination of IK blockade and PVI prevented AF in simulations without fibrosis, with moderate fibrosis, and even with severe fibrosis. The combined therapy strongly decreased the number of fibrillation waves, due to a synergistic reduction of wavefront generation rate while the wavefront lifespan remained unchanged. CONCLUSION: Newly developed blockers of atrial-specific inward rectifier currents, such as IKAch, might prevent AF occurrences and when combined with PVI effectively supress AF recurrences in human.

The Acetylcholine-Activated Potassium Current Inhibitor XAF-1407 Terminates Persistent Atrial Fibrillation in Goats.

Aims: The acetylcholine-activated inward rectifier potassium current (IKACh) has been proposed as an atrial-selective target for the treatment of atrial fibrillation (AF). Using a novel selective IKACh inhibitor XAF-1407, the study investigates the effect of IKACh inhibition in goats with pacing-induced, short-term AF. Methods: Ten goats (57 ± 5 kg) were instrumented with pericardial electrodes. Electrophysiological parameters were assessed at baseline and during intravenous infusion of XAF-1407 (0.3, 3.0 mg/kg) in conscious animals before and after 2 days of electrically induced AF. Following a further 2 weeks of sustained AF, cardioversion was attempted with either XAF-1407 (0.3 followed by 3 mg/kg) or with vernakalant (3.7 followed by 4.5 mg/kg), an antiarrhythmic drug that inhibits the fast sodium current and several potassium currents. During a final open chest experiment, 249 unipolar electrograms were recorded on each atrium to construct activation patterns and AF cardioversion was attempted with XAF-1407. Results: XAF-1407 prolonged atrial effective refractory period by 36 ms (45%) and 71 ms (87%) (0.3 and 3.0 mg/kg, respectively; pacing cycle length 400 ms, 2 days of AF-induced remodeling) and showed higher cardioversion efficacy than vernakalant (8/9 vs. 5/9). XAF-1407 caused a minor decrease in the number of waves per AF cycle in the last seconds prior to cardioversion. Administration of XAF-1407 was associated with a modest increase in QTc (<10%). No ventricular proarrhythmic events were observed. Conclusion: XAF-1407 showed an antiarrhythmic effect in a goat model of AF. The study indicates that IKACh represents an interesting therapeutic target for treatment of AF. To assess the efficacy of XAF-1407 in later time points of AF-induced remodeling, follow-up studies with longer period of AF maintenance would be necessary.

Intravenous administration of normal saline may be misinterpreted as a change of end-expiratory lung volume when using electrical impedance tomography.

Electrical impedance tomography (EIT) is a noninvasive imaging modality that allows real-time monitoring of regional lung ventilation. The aim of the study is to investigate whether fast saline infusion causes changes in lung impedance that could affect the interpretation of EIT data. Eleven pigs were anaesthetized and mechanically ventilated. A bolus of 500 mL of normal saline was administered rapidly. Two PEEP steps were performed to allow quantification of the effect of normal saline on lung impedance. The mean change of end-expiratory lung impedance (EELI) caused by the saline bolus was equivalent to a virtual decrease of end-expiratory lung volume (EELV) by 227 (188-250) mL and decremental PEEP step of 4.40 (3.95-4.59) cmH2O (median and interquartile range). In contrast to the changes of PEEP, the administration of normal saline did not cause any significant differences in measured EELV, regional distribution of lung ventilation determined by EIT or in extravascular lung water and intrathoracic blood volume. In conclusion, EELI can be affected by the changes of EELV as well as by the administration of normal saline. These two phenomena can be distinguished by analysis of regional distribution of lung ventilation.