Porcine Model Of Atrial Fibrillation Research Articles

Comparison of the antiarrhythmic potential of doxapram and its metabolite ketodoxaparam

Abstract Background A few years ago, the TASK-1 channel has been established as a potential new target for the therapy of atrial fibrillation (AF). In the heart, TASK-1 is almost exclusively expressed in the atria and is significantly upregulated in AF patients. Therefore, it plays an important role in the shortening of the atrial action potential observed during AF, making TASK-1 a promising target for AF therapy. This could be proven in a porcine model of persistent AF in which an intravenous application of the TASK-1 inhibitor doxapram led to a termination of AF and the restoration of sinus rhythm (SR). Doxapram's metabolite ketodoxapam is described in the literature as being active with limited data available on its usage. Therefore, the effect of ketodoxapram on TASK-1 and its possible use in AF therapy still needs to be investigated. Purpose The purpose of the present study was to assess the potential of ketodoxapram in the termination of AF. Furthermore, a comparison between doxapram and ketodoxapram was performed based on electrophysiological and pharmacological data. Methods UPLC-MS/MS assays were developed and validated for the measurement of doxapram and ketodoxapram in porcine plasma and brain tissue. Using these assays, the pharmacokinetics of both substances, after intravenous injections of 1 mg/kg, were determined in pigs. Furthermore, brain and plasma concentrations were measured to assess brain-to-plasma ratios. A porcine AF model was used to estimate the antiarrhythmic potential. Electrophysiological properties were evaluated, using two-electrode voltage clamp experiment on Xenopus laevis oocytes which heterologously expressed atrial potassium channels, to calculate the effect of doxapram and ketodoxapram on channel function. Furthermore, whole-cell patch clamp measurements were performed on isolated human cardiomyocytes. Results Doxapram and ketodoxapram showed strong inhibitory effects on TASK-1 (IC50 1.0 μM and 0.8 μM) and TASK-3 (5.9 μM and 1.5 μM), but no significant effect on the other measured ion channels. The maximal inhibition on TASK-1 was 96%. The brain-to-plasma ratio for doxapram (0.58) was almost ten-fold higher than for ketodoxapram (0.065), hinting at a reduced crossing of the blood-brain barrier (BBB) for ketodoxapram. The terminal half-life (t1/2) of ketodoxapram (1.71 h) was longer and the maximal concentration (Cmax; 4,604 ng/ml) was higher than for doxapram (1.38 h; 1,780 ng/ml). In a porcine model of AF, ketodoxapram led to a significantly reduced AF burdens comparable to doxapram. Conclusions Doxapram and ketodoxapram both show strong inhibitory effects on TASK-1, making them good candidates for a TASK-1 based AF therapy. Ketodoxapram with its longer t1/2, reduced crossing of the BBB and higher Cmax points towards a possible superiority in the treatment of AF compared to doxapram. Studies in a porcine AF model showed promising results for the use of doxapram and ketodoxapram in AF therapy. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Research Foundation

Treatment of atrial fibrillation with doxapram: TASK-1 potassium channel inhibition as a novel pharmacological strategy.

TASK-1 (K2P3.1) two-pore-domain potassium channels are atrial-specific and significantly up-regulated in atrial fibrillation (AF) patients, contributing to AF-related electrical remodelling. Inhibition of TASK-1 in cardiomyocytes of AF patients was shown to counteract AF-related action potential duration shortening. Doxapram was identified as a potent inhibitor of the TASK-1 channel. In this study, we investigated the antiarrhythmic efficacy of doxapram in a porcine model of AF. Doxapram successfully cardioverted pigs with artificially induced episodes of AF. We established a porcine model of persistent AF in domestic pigs via intermittent atrial burst stimulation using implanted pacemakers. All pigs underwent catheter-based electrophysiological investigations prior to and after 14 days of doxapram treatment. Pigs in the treatment group received intravenous administration of doxapram once per day. In doxapram-treated AF pigs, the AF burden was significantly reduced. After 14 days of treatment with doxapram, TASK-1 currents were still similar to values of sinus rhythm animals. Doxapram significantly suppressed AF episodes and normalized cellular electrophysiology by inhibition of the TASK-1 channel. Patch-clamp experiments on human atrial cardiomyocytes, isolated from patients with and without AF could reproduce the TASK-1 inhibitory effect of doxapram. Repurposing doxapram might yield a promising new antiarrhythmic drug to treat AF in patients.

Current Drug Treatment Strategies for Atrial Fibrillation and TASK-1 Inhibition as an Emerging Novel Therapy Option.

Atrial fibrillation (AF) is the most common sustained arrhythmia with a prevalence of up to 4% and an upwards trend due to demographic changes. It is associated with an increase in mortality and stroke incidences. While stroke risk can be significantly reduced through anticoagulant therapy, adequate treatment of other AF related symptoms remains an unmet medical need in many cases. Two main treatment strategies are available: rate control that modulates ventricular heart rate and prevents tachymyopathy as well as rhythm control that aims to restore and sustain sinus rhythm. Rate control can be achieved through drugs or ablation of the atrioventricular node, rendering the patient pacemaker-dependent. For rhythm control electrical cardioversion and pharmacological cardioversion can be used. While electrical cardioversion requires fasting and sedation of the patient, antiarrhythmic drugs have other limitations. Most antiarrhythmic drugs carry a risk for pro-arrhythmic effects and are contraindicated in patients with structural heart diseases. Furthermore, catheter ablation of pulmonary veins can be performed with its risk of intraprocedural complications and varying success. In recent years TASK-1 has been introduced as a new target for AF therapy. Upregulation of TASK-1 in AF patients contributes to prolongation of the action potential duration. In a porcine model of AF, TASK-1 inhibition by gene therapy or pharmacological compounds induced cardioversion to sinus rhythm. The DOxapram Conversion TO Sinus rhythm (DOCTOS)-Trial will reveal whether doxapram, a potent TASK-1 inhibitor, can be used for acute cardioversion of persistent and paroxysmal AF in patients, potentially leading to a new treatment option for AF.

Fast delivery of flecainide via the pulmonary (bolus) or intravenous (rapid infusion) routes reduces atrial fibrillation conversion dose and minimizes negative inotropic burden

Abstract Background/Introduction The negative inotropic effect of certain antiarrhythmic drugs limits their use for acute cardioversion of new-onset paroxysmal atrial fibrillation (AF). Purpose In an intact porcine model of AF, we examined the effects of pulmonary and intravenous (IV) administration of flecainide on left ventricular (LV) contractility, i.e., LV dP/dt max, at doses that are effective in converting AF to sinus rhythm. The magnitude of the decrease in LV dP/dt max and time that it remained below baseline, measured by the area under the curve (AUC), is referred to as negative inotropic burden. Methods Flecainide was delivered via intratracheal administration at 1.5 mg/kg bolus and compared to IV infusion at 1.0 mg/kg over 2 min (lower-dose, rapid) and 2.0 mg/kg over 10 min (ESC guideline) in 11 closed-chest, anesthetized Yorkshire pigs. These doses of flecainide have been shown effective in converting AF to sinus rhythm. Catheters were fluoroscopically positioned in the right atrium for pacing at 140 beats/min and in the LV to measure QRS complex duration and contractility (LV dP/dt). Intratracheal flecainide was delivered via a catheter positioned at the bifurcation of the main bronchi. Results The peak plasma levels (Cmax values) were similar but the AUC of plasma concentrations over time was greater for the higher-dose, slow IV infusion of flecainide than for either intratracheal instillation (by 32%) or lower-dose, rapid IV infusion (by 88%). Based on AUCs of LV dP/dt max (figure, left panel), the negative inotropic burden is 3.1- to 3.8-fold greater for the higher IV (1006% • min) than for the lower IV (323%·min) or the intratracheal doses (263% • min). There was a corresponding inverse increase in the AUC of QRS complex prolongation. The decrease in LV dP/dt max (Δ%) was correlated with the prolongation of the QRS complex (Δms) (y = −1.4337x + 3.6613, r2=0.69, p<0.0001) (figure, right panel). Conclusion Rapid delivery of pulmonary or IV flecainide reduces the dose of drug required to achieve Cmax levels associated with conversion of AF. The attendant decrease across time in exposure of the ventricles to flecainide reduces QRS complex prolongation and the accompanying negative inotropic burden. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): InCarda Therapeutics, Inc.

Characterization of Atrial and Ventricular Structural Remodeling in a Porcine Model of Atrial Fibrillation Induced by Atrial Tachypacing.

Background: Atrial fibrillation (AF) is characterized by electrical and structural remodeling. Irregular and/or fast atrio-ventricular (AV) conduction during AF can result in AV dyssynchrony, tachymyopathy, pressure and volume overload with subsequent dilatation, valve regurgitation, and ventricular dysfunction with progression to heart failure.Objective: To gain further insight into the myocardial pathophysiological changes induced by right atrial tachypacing (A-TP) in a large animal model.Methods: A total of 28 Landrace pigs were randomized as 14 into AF-induced A-TP group and 14 pigs to control group. AF pigs were tachypaced for 43 ± 4 days until in sustained AF. Functional remodeling was investigated by echocardiography (after cardioversion to sinus rhythm). Structural remodeling was quantified by histological preparations with picrosirius red and immunohistochemical stainings.Results: A-TP resulted in decreased left ventricular ejection fraction (LVEF) accompanied by increased end-diastolic and end-systolic left atrium (LA) volume and area. In addition, A-TP was associated with mitral valve (MV) regurgitation, diastolic dysfunction and increased atrial and ventricular fibrotic extracellular matrix (ECM).Conclusions: A-TP induced AF with concomitant LV systolic and diastolic dysfunction, increased LA volume and area, and atrial and ventricular fibrosis.

1206Doxapram is a promising new antiarrhythmic drug for an atrial-specific therapy of atrial fibrillation

Abstract Background TASK-1 (K2P3.1) is an atrial-specific two-pore domain potassium channel that is significantly upregulated in atrial fibrillation (AF) patients resulting in shortened atrial action potential duration (APD). Inhibition of TASK-1 in human atrial cardiomyocytes reverses AF-related APD shortening to values observed in patients with sinus rhythm (SR). By in silico-modelling and experimental characterization of drug binding sites, doxapram was identified as specific inhibitor of TASK-1. Purpose In this study, we investigated the antiarrhythmic efficacy of doxapram in a porcine model of AF to convert and suppress AF. Methods We established a new porcine model of persistent AF without induced tachymyopathy. AF was induced in domestic pigs by intermittent atrial burst stimulation using implanted pacemakers. During AF episodes, burst stimulation was inhibited by an integrated pacemaker biofeedback algorithm. AV-node ablation was performed to prevent AF-associated heart failure. All pigs underwent catheter-based electrophysiological investigations prior to and after 14 days doxapram treatment. Pigs in the treatment group received intravenous applications of doxapram twice per day. Rhythm status was continuously recorded by intracardiac long-term ECG monitors. The application of doxapram for cardioversion and long term suppression of AF in pigs with persistent AF was evaluated. Subsequent to the doxapram treatment, porcine cardiomyocytes were isolated from right and left atria and electrophysiologically investigated by patch-clamp and multi-electrode experiments. Atrial electrical remodeling was characterized by analyses of ion channel expression at mRNA and protein levels. Results TASK-1 mRNA, protein and transmembrane current were significantly increased in AF pigs compared to SR controls, resulting in shortened atrial APDs. In doxapram-treated AF pigs the AF burden was significantly reduced. After 14 days treatment with doxapram, TASK-1 currents and atrial APDs recorded in porcine cardiomyocytes were reduced and similar to values of SR animals. Doxapram could be successfully applied for cardioversion in pigs with persistent AF. On average, cardioversion was observed 3 minutes after doxapram application. Conclusion Doxapram significantly suppressed AF episodes and normalized cellular electrophysiological characteristics in a porcine model of AF through inhibition of the TASK-1 ion channel. Furthermore, doxapram rapidly converted AF into SR in pigs. Therefore, doxapram might serve as a new antiarrhythmic drug to treat AF in patients.

Genetic Ablation of TASK-1 (Tandem of P Domains in a Weak Inward Rectifying K+ Channel-Related Acid-Sensitive K+ Channel-1) (K2P3.1) K+ Channels Suppresses Atrial Fibrillation and Prevents Electrical Remodeling.

Despite an increasing understanding of atrial fibrillation (AF) pathophysiology, translation into mechanism-based treatment options is lacking. In atrial cardiomyocytes of patients with chronic AF, expression, and function of tandem of P domains in a weak inward rectifying TASK-1 (K+ channel-related acid-sensitive K+ channel-1) (K2P3.1) atrial-specific 2-pore domain potassium channels is enhanced, resulting in action potential duration shortening. TASK-1 channel inhibition prevents action potential duration shortening to maintain values observed among sinus rhythm subjects. The present preclinical study used a porcine AF model to evaluate the antiarrhythmic efficacy of TASK-1 inhibition by adeno-associated viral anti-TASK-1-siRNA (small interfering RNA) gene transfer. AF was induced in domestic pigs by atrial burst stimulation via implanted pacemakers. Adeno-associated viral vectors carrying anti-TASK-1-siRNA were injected into both atria to suppress TASK-1 channel expression. After the 14-day follow-up period, porcine cardiomyocytes were isolated from right and left atrium, followed by electrophysiological and molecular characterization. AF was associated with increased TASK-1 transcript, protein and ion current levels leading to shortened action potential duration in atrial cardiomyocytes compared to sinus rhythm controls, similar to previous findings in humans. Anti-TASK-1 adeno-associated viral application significantly reduced AF burden in comparison to untreated AF pigs. Antiarrhythmic effects of anti-TASK-1-siRNA were associated with reduction of TASK-1 currents and prolongation of action potential durations in atrial cardiomyocytes to sinus rhythm values. Conclusions Adeno-associated viral-based anti-TASK-1 gene therapy suppressed AF and corrected cellular electrophysiological remodeling in a porcine model of AF. Suppression of AF through selective reduction of TASK-1 currents represents a new option for antiarrhythmic therapy.

Arterial hypertension drives arrhythmia progression via specific structural remodeling in a porcine model of atrial fibrillation

Arterial hypertension (HT) contributes to progression of atrial fibrillation (AF) via unknown mechanisms. We aimed to characterize electrical and structural changes accounting for increased AF stability in a large animal model of rapid atrial pacing (RAP)-induced AF combined with desoxycorticosterone acetate (DOCA)-induced HT. Eighteen pigs were instrumented with right atrial endocardial pacemaker leads and custom-made pacemakers to induce AF by continuous RAP (600 beats/min). DOCA pellets were subcutaneously implanted in a subgroup of 9 animals (AF+HT group); the other 9 animals served as controls (AF group). Final experiments included electrophysiology studies, endocardial electroanatomic mapping, and high-density mapping with epicardial multielectrode arrays. In addition, 3-dimensional computational modeling was performed. DOCA implantation led to secondary HT (median [interquartile range] aortic pressure 109.9 [100-137] mm Hg in AF+HT vs 82.2 [79-96] mm Hg in AF; P < .05), increased AF stability (55.6% vs 12.5% of animals with AF episodes lasting >1 hour; P < .05), concentric left ventricular hypertrophy, atrial dilatation (119 ± 31 cm2 in AF+HT vs 78 ± 23 cm2 in AF; P<.05), and fibrosis. Collagen accumulation in the AF+HT group was mainly found in non-intermyocyte areas (1.62 ± 0.38 cm3 in AF+HT vs 0.96 ± 0.3 cm3 in AF; P < .05). Left and right atrial effective refractory periods, action potential durations, endo- and epicardial conduction velocities, and measures of AF complexity were comparable between the 2 groups. A 3-dimensional computational model confirmed an increase in AF stability observed in the invivo experiments associated with increased atrial size. In this model of secondary HT, higher AF stability after 2 weeks of RAP is mainly driven by atrial dilatation.

Atrial myofibroblast activation and connective tissue formation in a porcine model of atrial fibrillation and reduced left ventricular function

AimsAtrial fibrillation (AF) is associated with fibrosis that slows electrical conduction and causes perpetuation of the arrhythmia. The molecular characterization of AF pathophysiology may provide novel therapeutic options. This study was designed to elucidate profibrotic signaling and myofibroblast activation in a porcine model of atrial tachypacing-induced AF and reduced left ventricular function. Materials and methodsTen domestic pigs were randomized to sinus rhythm (SR) or AF groups. Prior to AF induction and on day 14 the animals underwent echocardiographic examinations. Profibrotic pathways were analyzed in right atrial tissue obtained from AF animals compared to SR controls using histology, immunofluorescence microscopy, Western blot, and real-time PCR. Key findingsAF was associated with atrial dilation, increased atrial fibrosis, and enhanced expression of collagens I and V in right atrial tissue after 14days follow-up. The fraction of α-smooth muscle actin (SMA)-producing activated myofibroblasts was elevated in AF, whereas the abundance of vimentin-expressing inactive fibroblasts was not affected. Profibrotic signaling involved upregulation of TGF-β1, Smad2/3, and CTGF. SignificanceThe transformation of atrial fibroblasts into myofibroblasts through activation of TGF-β1 and CTGF emerged as potential cellular trigger of fibrogenesis. Prevention of fibroblast-to-myofibroblast switching may serve as target for remodeling-based antiarrhythmic AF therapy.

The impact of 6 weeks of atrial fibrillation on left atrial and ventricular structure and function

ObjectiveThe impact of prolonged episodes of atrial fibrillation on atrial and ventricular function has been incompletely characterized. The purpose of this study was to investigate the influence of atrial fibrillation on left atrial and ventricular function in a rapid paced porcine model of atrial fibrillation. MethodsA control group of pigs (group 1, n = 8) underwent left atrial and left ventricular conductance catheter studies and fibrosis analysis. A second group (group 2, n = 8) received a baseline cardiac magnetic resonance imaging to characterize left atrial and left ventricular function. The atria were rapidly paced into atrial fibrillation for 6 weeks followed by cardioversion and cardiac magnetic resonance imaging. ResultsAfter 6 weeks of atrial fibrillation, left atrial contractility defined by atrial end-systolic pressure-volume relationship slope was significantly lower in group 2 than in group 1 (1.1 ± 0.5 vs 1.7 ± 1.0; P = .041), whereas compliance from the end-diastolic pressure-volume relationship was unchanged (1.5 ± 0.9 vs 1.6 ± 1.3; P = .733). Compared with baseline, atrial fibrillation resulted in a significantly higher contribution of left atrial reservoir volume to stroke volume (32% vs 17%; P = .005) and lower left atrial booster pump volume contribution to stroke volume (19% vs 28%; P = .029). Atrial fibrillation also significantly increased maximum left atrial volume (206 ± 41 mL vs 90 ± 21 mL; P < .001). Left atrial fibrosis in group 2 was significantly higher than in group 1. Atrial fibrillation decreased left ventricular ejection fraction (29% ± 9% vs 58 ± 8%; P < .001), but left ventricular stroke volume was unchanged. ConclusionsIn a chronic model of atrial fibrillation, the left atrium demonstrated significant structural remodeling and decreased contractility. These data suggest that early intervention in patients with persistent atrial fibrillation might mitigate against adverse atrial and ventricular structural remodeling.

Cloning, functional characterization, and remodeling of K2P3.1 (TASK-1) potassium channels in a porcine model of atrial fibrillation and heart failure.

Effective treatment of atrial fibrillation (AF) remains an unmet need. Human K2P3.1 (TASK-1) K(+) channels display atrial-specific expression and may serve as novel antiarrhythmic targets. In rodents, inhibition of K2P3.1 causes prolongation of action potentials and QT intervals. We used a porcine model to further elucidate the significance of K2P3.1 in large mammals. The purpose of this study was to study porcine (p)K2P3.1 channel function and cardiac expression and to analyze pK2P3.1 remodeling in AF and heart failure (HF). The porcine K2P3.1 ortholog was amplified and characterized using voltage-clamp electrophysiology. K2P3.1 mRNA expression and remodeling were studied in domestic pigs during AF and HF induced by atrial burst pacing. Porcine K2P3.1 cDNA encodes a channel protein with 97% identity to human K2P3.1. K(+) currents recorded from Xenopus oocytes expressing pK2P3.1 were functionally and pharmacologically similar to their human counterparts. In the pig, K2P3.1 mRNA was predominantly expressed in atrial tissue. AF and HF were associated with reduction of K2P3.1 mRNA levels by 85.1% (right atrium) and 77.0% (left atrium) at 21-day follow-up. In contrast, ventricular K2P3.1 expression was low and not significantly affected by AF/HF. Porcine K2P3.1 channels exhibit atrial expression and functional properties similar to their human orthologs, supporting a general role as antiarrhythmic drug targets. K2P3.1 down-regulation in AF with HF may indicate functional relevance of the channel that remains to be validated in prospective interventional studies.