Goat Model Of Atrial Fibrillation Research Articles

Compartmentalization proteomics revealed endolysosomal protein network changes in a goat model of atrial fibrillation

Endolysosomes (EL) are known for their role in regulating both intracellular trafficking and proteostasis. EL facilitate the elimination of damaged membranes, protein aggregates, membranous organelles and play an important role in calcium signaling. The specific role of EL in cardiac atrial fibrillation (AF) is not well understood. We isolated atrial EL organelles from AF goat biopsies and conducted a comprehensive integrated omics analysis to study the EL-specific proteins and pathways. We also performed electron tomography, protein and enzyme assays on these biopsies. Our results revealed the upregulation of the AMPK pathway and the expression of EL-specific proteins that were not found in whole tissue lysates, including GAA, DYNLRB1, CLTB, SIRT3, CCT2, and muscle-specific HSPB2. We also observed structural anomalies, such as autophagic-vacuole formation, irregularly shaped mitochondria, and glycogen deposition. Our results provide molecular information suggesting EL play a role in AF disease process over extended time frames.

High-density sequential mapping of repetitive atrial conduction patterns during atrial fibrillation

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): This work was supported by PersonalizeAF project. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 860974. Background Localized AF drivers are considered candidate ablation targets for patients with persistent atrial fibrillation (AF). These drivers are expected to be associated with repetitive atrial conduction patterns during AF. Thus, tools that localize atrial sites with repetitive electrical activity might be instrumental in guiding ablation. Purpose High-density mapping catheters cover only a small portion of the atria. Combining sequential recordings from those catheters could provide a more complete picture of repetitive conduction patterns, and enable AF driver localization. We hypothesize that the repetitive activity generated by local AF drivers can be detected by means of high-coverage composite activation maps generated from spatially overlapping sequential recordings. Methods Repetitive conduction patterns were detected in a goat model of AF (249-electrode epicardial mapping array, 2.4mm inter-electrode distance, n=16) by exploiting recurrence plots (Fig 1A-C). Cross-recurrences of repetitive patterns in sequential recordings were detected in spatially overlapping recording locations. Using this information, local activation maps were aligned and combined into larger composite average activation maps (Fig. 1D-F). The proposed algorithm was tested on a dataset formed by segmenting the epicardial mapping area into four spatially overlapping regions. The proposed algorithm was then used to merge these segmented regions back together to reconstruct the original mapping area. Reconstruction accuracy was measured as the correlation between original and reconstructed average activation patterns (Fig. 2.). Statistical analyses were performed to investigate a possible relation between reconstruction accuracy and pattern properties such as duration, size, complexity, and cycle length. Patterns were classified as single peripheral, multiple waves, focal source, or re-entry based on the preferential conduction velocity directions. Results Among 1021 detected repetitive patterns, 328 spatiotemporally stable- patterns were present in all four artificially segmented recordings. In 32% of these, repetitiveness was associated with a local driver-either focal or re-entrant. Composite maps could be generated in 75% of the cases, and mainly in case of larger patterns (p<0.01). The average correlation between the actual activation maps and the composite maps was 0.86 ±0.16. Only pattern duration showed a statistically significant low correlation with reconstruction accuracy of composite maps (r=0.126, p<0.05). There was no significant difference in the reconstruction accuracy for multiple waves, focal sources and re-entries. Conclusion(s) The proposed framework could align sequentially recorded repetitive epicardial patterns over different atrial regions, to produce high-fidelity composite maps. The performance was minimally affected by pattern properties, thus suggesting potential use with a diverse range of AF patterns.

Thrombin generation by calibrated automated thrombography in goat plasma: Optimization of an assay

The goat model of atrial fibrillation (AF) allows investigation of the effect of AF on coagulation. However, assays for goat plasma are not available from commercial sources. Calibrated automated thrombography (CAT) provides a global view of the coagulation profile by assessing in vitro thrombin generation (TG). We describe the customization of the CAT assay in goat platelet‐poor plasma (PPP) and in factor Xa (FXa)‐inhibitor‐anticoagulated PPP. TG was initiated in the presence of phospholipids and either (a) PPP reagent, reagent low, or reagent high; (b) goat brain protein extraction (GBP); or (c) Russell's viper venom‐factor X activator (RVV‐X). Contact activation was assessed by adding corn trypsin inhibitor. Different concentrations of prothrombin complex concentrate (PCC) were used to determine the sensitivity of both the GBP and RVV‐X method. To obtain FXa‐inhibitor anticoagulated plasma, rivaroxaban was added to plasma. TG settings with human reagents were not suitable for goat plasma. TG triggered with GBP increased peak height and ETP values. Similarly, the RVV‐X method produced comparable TG curves and was more sensitive to PCC titration. Finally, both methods were able to detect the decrease in clotting potential induced by FXa inhibition. This is the first study that reports the customization of the CAT assay for goats. The GBP and RVV‐X methods were comparable in triggering TG in goat plasma. The RVV‐X method seemed to better discriminate changes in TG curves due to increases in clotting potential as well as to FXa inhibition by rivaroxaban in goat plasma.

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.

JavaCyte, a novel open-source tool for automated quantification of key hallmarks of cardiac structural remodeling

Many cardiac pathologies involve changes in tissue structure. Conventional analysis of structural features is extremely time-consuming and subject to observer bias. The possibility to determine spatial interrelations between these features is often not fully exploited. We developed a staining protocol and an ImageJ-based tool (JavaCyte) for automated histological analysis of cardiac structure, including quantification of cardiomyocyte size, overall and endomysial fibrosis, spatial patterns of endomysial fibrosis, fibroblast density, capillary density and capillary size. This automated analysis was compared to manual quantification in several well-characterized goat models of atrial fibrillation (AF). In addition, we tested inter-observer variability in atrial biopsies from the CATCH-ME consortium atrial tissue bank, with patients stratified by their cardiovascular risk profile for structural remodeling. We were able to reproduce previous manually derived histological findings in goat models for AF and AV block (AVB) using JavaCyte. Furthermore, strong correlation was found between manual and automated observations for myocyte count (r = 0.94, p < 0.001), myocyte diameter (r = 0.97, p < 0.001), endomysial fibrosis (r = 0.98, p < 0.001) and capillary count (r = 0.95, p < 0.001) in human biopsies. No significant variation between observers was observed (ICC = 0.89, p < 0.001). We developed and validated an open-source tool for high-throughput, automated histological analysis of cardiac tissue properties. JavaCyte was as accurate as manual measurements, with less inter-observer variability and faster throughput.

Hypercoagulability Promotes Atrial Fibrosis and Fibrillation

Background: It is well known that atrial fibrillation (AF) induces a hypercoagulable state, which significantly increases stroke risk in patients with AF contributing to morbidity and mortality in these patients. Active coagulation factors can also provoke diverse cellular responses through stimulation of protease-activated receptors (PARs). In the heart and vessels, coagulation factor mediated PAR activation may provoke and mediate pro-inflammatory and tissue remodeling responses, potentially contributing to organ damage. We hypothesized that the onset and progression of AF, may be affected by hypercoagulability-mediated cell signaling responses, in the heart.Methods and results: To study the potential role of PARs in the structural remodeling process that renders the atria more prone to AF we first investigated whether thrombin or factor Xa could induce atrial fibroblast remodeling. In isolated rat cardiac fibroblasts, thrombin enhanced the phosphorylation of the pro-fibrotic signaling molecules Akt and Erk, and increased expression of TGFβ1 (2.7 fold) and the pro-inflammatory factor monocyte chemo-attractant protein-1 (6.1 fold). Thrombin also increased the incorporation of 3H-proline suggesting enhanced collagen synthesis by cardiac fibroblasts (2.5 fold). Differentiation towards myofibroblasts was indicated by increased expression of smooth muscle actin (2 fold). All effects could be prevented by the direct thrombin inhibitor dabigatran and comparable results were obtained for stimulation with factor Xa and inhibition with rivaroxaban, respectively.Next we studied whether enhanced stimulation of PARs by chronic elevation of thrombin levels would lead to an enhanced vulnerability to AF in transgenic mice. In mice with enhanced thrombin activity due to a mutation in the thrombomodulin gene resulting in impaired thrombin inhibition (TMpro/pro), inducibility of AF episodes provoked by burst pacing was higher (6 out of 10 versus 1 out of 10 in wild type) and the duration of AF episodes was longer (episodes >2s in 6 out of 10 versus 0 out of 10 in wild type). Finally, we showed that inhibition of the coagulation cascade attenuated the development of AF in a goat model of AF. In 6 goats with persistent AF and treated with the anticoagulant nadroparine (4 weeks, 150 IU/kg twice daily) the complexity of the AF substrate was less pronounced compared to control animals. The conduction heterogeneity and block were 33% shorter in the nadroparine treated animals (maximal conduction time 23.3±3.1ms in control versus 15.7±2.1ms in nadroparine, p<0.05) and AF-induced a-SMA expression and endomysial fibrosis were less pronounced.Conclusion: The hypercoagulable state during AF provokes pro-fibrotic and pro-inflammatory responses in cardiac fibroblasts, as well as promotes the development of a substrate for AF in transgenic mice and in goats with persistent AF. Together, these results strongly support the role of hypercoagulability and PAR activation in the development of a substrate for AF. In addition, direct anticoagulant treatment may protect against AF-related cellular atrial remodelling. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Loss of Continuity in the Thin Epicardial Layer Because of Endomysial Fibrosis Increases the Complexity of Atrial Fibrillatory Conduction

Background— The transition from persistent to permanent atrial fibrillation (AF) is associated with increased complexity of fibrillatory conduction. We have investigated the spatial distribution of fibrillation waves and structural alterations in the atrial free walls in a goat model of AF. Methods and Results— AF was maintained for 3 weeks (short term [ST], persistent AF) or 6 months (long term [LT], permanent AF). Fibrillation patterns were assessed with epicardial mapping. The origin of fibrillation waves and sites of conduction abnormalities were more homogeneously distributed in LT than in ST goats. Histologically, the total area fraction occupied by fibrous tissue and the degree of perimysial fibrosis (separation between myocyte bundles) were not significantly different between groups. However, endomysial fibrosis (distance between myocytes within bundles) was significantly larger in LT goats, particularly in the outer millimeter of the atria. By contrast, myocyte diameters were larger in LT goats throughout the atrial walls. High-resolution optical mapping showed that epicardial wavefront expansion was slower and more anisotropic in LT than in ST goats. Finally, a mathematical model of a simplified atrial architecture confirmed the potential impact of epicardial endomysial fibrosis on AF complexity. Conclusions— Altered propagation after 6 months of AF is consistent with homogeneous structural remodeling in the outer millimeter of the atria. Loss of continuity of the epicardial layer because of endomysial fibrosis may reduce its synchronizing effect, thereby increasing the complexity of fibrillatory conduction pathways. The exact distribution of fibrosis may be more important for the occurrence of conduction disturbances than the overall quantity.

AVE0118, Blocker of the Transient Outward Current ( I to ) and Ultrarapid Delayed Rectifier Current ( I Kur ), Fully Restores Atrial Contractility After Cardioversion of Atrial Fibrillation in the Goat

The loss of atrial contractile function after cardioversion of atrial fibrillation (AF) contributes to the thromboembolic risk associated with AF. The newly developed blocker of the transient outward current (I(to)) and ultrarapid delayed rectifier current (I(Kur)) AVE0118 prolongs atrial action potential duration and might therefore enhance atrial contractility. We compared the ability of AVE0118 to restore atrial contraction after cardioversion of AF with the efficacy of conventional positive inotropic compounds in the goat model of AF. Eighteen goats were chronically instrumented with epicardial electrodes, a pressure transducer in the right atrium, and piezoelectric crystals to measure right atrial diameter. Atrial contractility and refractoriness and QT duration were measured before and after 1 week (3 to 8 days) of AF induced by repetitive burst pacing. The measurements were repeated after administration of digoxin (0.02 mg/kg), dobutamine (5 microg x kg(-1) x min(-1)), the Ca2+ sensitizer EMD57033 (1 mg x kg(-1) x min(-1)), the L-type Ca2+ channel agonist BayY5959 (0.1 mg x kg(-1) x min(-1)), and AVE0118 (0.01 to 0.2 mg x kg(-1) x min(-1)). The effect of AVE0118 on the configuration of atrial monophasic action potentials was determined for comparison. After 1 week of AF, atrial contractility during sinus rhythm or slow atrial pacing was reduced to <10%. Digoxin and dobutamine failed to increase atrial contractility. EMD57033 restored 41% and BayY5959 restored 48% of atrial contractility at baseline. BayY5959 significantly prolonged QT duration by 24.7%. AVE0118 enhanced atrial contraction to 156% of the baseline value. The positive inotropic effect was accompanied by a pronounced prolongation of atrial action potential duration and refractoriness, whereas QT duration remained unchanged. Conventional positive inotropic drugs showed limited effect on atrial contractility after cardioversion of AF or produced QT prolongation. In contrast, the I(to)/I(Kur) blocker AVE0118 fully restored atrial contraction without proarrhythmic effects on the ventricle.

AB46-3: Conduction properties of Bachmann’s bundle in relation to tissue architecture in a goat model of atrial fibrillation

Bachmann’s bundle (BB) is a major conducting pathway between the left and right atrium (LA and RA). During atrial fibrillation (AF), electrograms recorded on BB display enhanced fragmentation and an increase of interatrial conduction delays. Beyond that, the conduction properties of BB and its possible role in AF perpetuation are largely unknown.

Profibrillatory effects of verapamil but not of digoxin in the goat model of atrial fibrillation.

Verapamil and digoxin have been shown to modulate tachycardia-induced atrial electrical remodeling. The goal of the present study was to determine the direct effects of verapamil and digoxin on atrial fibrillation (AF), before and after electrical remodeling. In six goats we measured the AF cycle length (AFCL) and duration of AF (DurAF) of 50 consecutive induced paroxysms, before (t = 0) and after 24 hours (t = 24) of electrical remodeling. During AF, conduction velocity (CV(AF)), refractory period (RP(AF)), and type of AF (I, II, III) were determined. Verapamil was administered at a loading dose of 0.1 mg/kg, followed by a continuous (2-hour) infusion of 5 microg/kg/min. Digoxin was given intravenously as a single 0.02 mg/kg bolus. At t = 0 and t = 24, digoxin and verapamil caused a significant slowing of the ventricular rate of >40%. Digoxin had no effect on DurAF, AFCL, CV(AF), or RP(AF). Infusion of verapamil had a direct proarrhythmic effect. Both at t = 0 and t = 24, AFCL and RP(AF) were shortened by about 15%. During acute AF, verapamil prolonged the average duration of AF paroxysms from 7 to 16 seconds. After 24 hours of AF, the proarrhythmic effect was much stronger. Shortly after starting infusion (6 +/- 2 min), verapamil converted paroxysmal AF into sustained AF. As long as verapamil infusion was maintained, AF no longer terminated in any of the goats. This effect was associated with an increase in AF fragmentation from type I to type II-III. Verapamil shortens AFCL and RP(AF) in the presence and absence of electrical remodeling. After 24 hours, it exerted a marked proarrhythmic effect and converted paroxysmal (type I) into sustained (type III) AF. In contrast, digoxin had no effect on the rate or stability of AF.