Rivaroxaban Treatment Prevents Atrial Myocyte Hypertrophy in Goats with Persistent Atrial Fibrillation by Inhibition of Protease-Activated Receptor-1

Abstract

Background

Atrial fibrillation (AF) is associated with an increased risk of stroke and hypercoagulability. Coagulation factors mediate remodeling processes via protease-activated receptors (PARs) in various organs.

Objective

We evaluated whether inhibition of factor Xa (FXa) via rivaroxaban protects against atrial structural remodeling in goats with persistent AF and explored FXa and thrombin hypertrophic effect on human iPSC-derived cardiomyocytes (hiPSC-CMs).

Methods

Three groups of goats were tested: CTRL AF (control AF, n = 10), RIVA AF (rivaroxaban treatment during AF, n = 11), and SHAM (no AF, n = 10). Pacing-induced AF was maintained for 16 weeks. AF stability, hemodynamics, and AF complexity were assessed. Atrial samples were collected for histological and gene expression analyses. hiPSC-CM were stimulated with PAR-1 agonist TRAP14, FXa, or thrombin with and without their inhibitors. Pro-hypertrophic and pro-inflammatory gene expression was assessed by qRT-PCR after 24 hours.

Results

Rivaroxaban inhibited thrombin generation in RIVA AF goats (baseline: 249 ± 42 nM vs. final: 69 ± 33 nM). Sixteen weeks of AF induced atrial myocyte hypertrophy in CTRL AF (13.5 µm [95% CI: 12.9, 14.0] vs. SHAM: 12.5 µm [95% CI: 12.0, 13.0]) and pro-hypertrophic (NPPA: fourfold; NPPB: 22-fold) and pro-fibrotic (COL1A1: threefold) gene expression. Rivaroxaban fully prevented hypertrophy (12.2 µm [95% CI: 11.7, 12.7]) and downregulated inflammatory signaling without altering hemodynamics and AF stability. In hiPSC-CM, thrombin and TRAP14 induced overexpression of the pro-hypertrophic genes NPPA and NPPB. The PAR1 antagonist, SCH79797, prevented thrombin-induced NPPA and NPPB upregulation.

Conclusion

Prolonged rivaroxaban treatment reduces thrombin generation, preventing AF-induced atrial myocyte hypertrophy through inhibition of PAR-1 signaling.

Keywords

atrial fibrillation - activated coagulation factors - protease-activated receptor-1 - atrial myocyte hypertrophy - rivaroxaban

Introduction

Atrial fibrillation (AF) is the most common chronic arrhythmia in clinical practice, affecting around 3% of the adult population and increasing the risk of thromboembolic stroke by fivefold.[1] [2] [3] [4] [5] [6] Patients with AF exhibit a hypercoagulable state, characterized by increased platelet activation, elevated levels of pro-thrombotic markers (e.g., prothrombin fragments 1 + 2 and thrombin-antithrombin complexes), and altered fibrinolytic activity.[6] [7] [8] [9] [10]

Coagulation factors like thrombin and factor Xa (FXa) have been shown to contribute to cardiac remodeling processes through the activation of protease-activated receptors (PARs).[11] [12] [13] [14] The PAR family consists of four isoforms (PAR-1 to -4), expressed in various cell types and organs, including the heart.[15] Previously, we showed that inhibition of FXa and thrombin by the low molecular weight heparin, nadroparin, reduced atrial fibrosis in goats with 4 weeks of pacing-induced AF. Furthermore, we found that thrombin has pro-fibrotic effects on cardiac fibroblasts mediated through PAR-1 signaling.[11] [12]

Mouse studies suggest that PAR-1 activation promotes ventricular hypertrophy.[16] [17] Importantly, hypertrophic cardiomyocytes may cause conduction disturbances and reduced conduction velocity within the atria, potentially contributing to an AF substrate.[18] [19] [20]

Direct oral anticoagulants (DOACs) selectively inhibit the activity of FXa and thrombin, reducing PAR activation.[21] [22] As a result, DOACs, like rivaroxaban and dabigatran, might not only prevent thromboembolic events but also protect against cardiac remodeling processes mediated by activated coagulation factors.

In this study, we hypothesize that, in goats with AF, long-term inhibition of FXa-mediated thrombin generation via rivaroxaban can: (1) attenuate atrial pro-fibrotic, pro-hypertrophic, and proinflammatory signaling, (2) modulate atrial structural remodeling processes, and (3) delay the development of an electrophysiological substrate for AF.

Methods

An expanded Methods are available at ([Supplementary Material] available in the online version only).

Animal Experiments

All animal experiments followed the European and national guidelines and were approved by Maastricht University's ethics committees for animal experiments (DEC2014–025).[23]

Rivaroxaban Pharmacokinetics in Goats

Rivaroxaban administration route and dosage were determined by an extensive pharmacokinetic study in goats ([Supplementary Table S1], available in the online version only). A twice-daily oral dosage of 3 mg/kg resulted in rivaroxaban plasma concentrations that remained in the therapeutic range for more than 12 hours after each administration ([Supplementary Fig. S1C], available in the online version only) and was chosen for this study.

Fig. 1 Timeline of the goat study. The red arrows indicate the blood sampling moments, the green arrows indicate the (maximum) four cardioversion attempts, the black arrows mark key experimental procedures, and the blue arrow indicates the initiation of rivaroxaban treatment. CTRL AF, control atrial fibrillation; RIVA AF, rivaroxaban atrial fibrillation.

Goat Study: General

Three experimental groups were included: AF only (CTRL AF, n = 10), AF + rivaroxaban (RIVA AF, n = 11), and sham (SHAM, n = 10; [Fig. 1]). AF was maintained for 16 weeks by burst pacing, as described below. In the RIVA AF group, rivaroxaban treatment was initiated 1 week prior to AF induction. The SHAM group underwent the same operation procedures as the other groups, but AF was never induced.[23]

Electrode Implantation for AF Induction

Goats were anesthetized to allow the implantation of a custom-made patch of 10 electrodes on the pericardium above the left atrium. Additionally, two-electrode patches were sutured to the pericardium overlying the left ventricle and on the right ventricle. The wires of the electrodes were combined in one silicone tube that was tunneled under the skin and exteriorized at the back of the goat for pacing and electrogram recording, as previously described.[24] After 2 weeks of recovery from surgery, AF was induced by burst pacing (50 Hz, ≤10 mA) once sinus rhythm (SR) was detected for 1 second. Subsequently, a subcutaneously implanted pacemaker was used to maintain AF (50 Hz, four times threshold) until the end of the experiment at 16 weeks of AF.

Blood Sampling and Rivaroxaban Monitoring

Blood was sampled at up to eight time points to monitor rivaroxaban plasma levels over time in the RIVA AF group ([Supplementary Fig. S1D], available in the online version only). Samples for coagulation analysis were taken at time points pre-Riva, pre-AF, and at the final experiment (SHAM: ± 8 weeks after the implantation, CTRL AF and RIVA AF: ± 18 weeks after implantation; [Fig. 1]). Blood was drawn from the jugular vein, processed into platelet-poor plasma (PPP), and stored at −80°C.[25]

Thrombin Generation Assay

Thrombin generation (TG) was measured in goat plasma by means of the calibrated automated thrombography method (Thrombinoscope B.V.), as previously described.[26] This analysis provides a thrombin generation curve reflecting the ability of plasma to generate thrombin. The TG curve is characterized by different parameters: lag time (initiation phase), ETP (endogenous thrombin potential), peak height (highest thrombin concentration), and start tail (time needed for generated thrombin to be fully inactivated).[27]

Cardioversion Attempts

To assess the stability of AF, pharmacological cardioversion attempts were performed at 3, 5, 10, and 15 weeks of AF. A cardioversion attempt consisted of 90 minutes of flecainide administration or until SR occurred. Experimental endpoints were AF termination, doubling of the QRS-width, and ventricular arrhythmia. Cardioversion was considered successful if AF terminated during drug infusion or during a 2-hour wash-out period. If two consecutive attempts did not terminate AF, the subsequent cardioversion attempts were not performed.

Atrial and ventricular electrograms were continuously monitored and recorded. The difference in AF cycle length between the start of flecainide infusion (baseline) and during the last minute before cardioversion (peak, if cardioversion occurred), or at 90 minutes after flecainide was determined.

Final Goat Experiment

Goats were anesthetized, and aortic and left ventricular (LV) pressures were measured using a pressure-tip catheter inserted into the carotid artery. A Swan-Ganz catheter was inserted into the jugular vein and advanced to the pulmonary artery to determine cardiac output by thermodilution. The mean of five consecutive measurements was taken for analysis. In the CTRL AF and RIVA AF goats, pressures were measured during AF, while in SHAM animals, pressures were measured during SR.

Subsequently, a left-sided thoracotomy was performed to expose the heart for direct atrial contact mapping. Two custom-made, round, high-density mapping electrodes (249 electrodes, interelectrode distance = 2.4 mm) were placed on the free wall of the left and right atria. Local unipolar electrograms were recorded during AF as previously described.[28] AF-electrogram files were analyzed offline using custom-made analysis software (MATLAB 8.1, MathWorks).[29]

Tissues for histological and gene expression analyses were harvested following the atrial mapping procedure. All organ samples were snap frozen in liquid nitrogen and stored at −80°C. Tibial length was measured from the left hind leg.

Analysis of Atrial Structural Remodeling

Cryosections (7 µm thickness) were cut transmurally from left and right atrial free wall samples and stained with wheat germ agglutinin (WGA, ThermoFisher). The automated analysis software “JavaCyte” was used to assess endomysial fibrosis (inter-myocyte distance) and myocyte hypertrophy, as previously described.[30]

Stimulation of iPSC-CM with FXa and Thrombin

Human iPSC line (WTC11, GM25256, Coriell Institute) was differentiated to atrial-like CM as described by Hutschalik et al.[31]

Beating atrial-like iPSC-CM were exposed to either human purified thrombin (8 pM, Hyphen, Biomed), human purified FXa (50 nM, Hyphen, Biomed), PAR1 agonist (100 µM, TRAP14, Bachem), or PAR1 antagonist (1 µM, SCH79797, Tocris, Bioscience). FXa and thrombin were incubated with their respective inhibitors, rivaroxaban (400 ng/mL, Bayer) and dabigatran (350 ng/mL, Boehringer Ingelheim) for 10 minutes before addition to the cells. Effects on gene expression were determined at 24 hours, unless otherwise specified.

Human BNP Assay

Conditioned media of human iPSC-CM incubated with thrombin with and without SCH79797 and TRAP14 for 24 hours were collected and stored at −80°C for analysis. Human BNP ELISA was performed according to the manufacturer's instructions (Invitrogen).

Gene Expression Analysis

Goat atrial tissue gene expression levels were normalized for the housekeeping gene HPRT and expressed as fold changes compared with SHAM.

For the iPSC-CM, gene expression levels were normalized for Cyclophilin-A. Results are expressed as fold-change relative to control.

The sequences of the specific primers used are provided in [Supplementary Tables S2] and [S3] (available in the online version only).

Statistical Analysis

All data are expressed as means ± standard deviation (SD), unless stated otherwise. p-Values < 0.05 were considered to be significant. Software programs SPSS (version 26, IBM) and PRISM (version 9.0.0, GraphPad) were used to compute all statistics. Detailed description of the test used can be found at [Supplementary Material] (available in the online version only).

Results

Physical Characteristics

In total, 31 female adult goats were used. The three study groups were comparable for age, body weight, and height (tibia length). Sixteen weeks of AF, with and without rivaroxaban treatment, did not affect heart, lung, kidney, or liver weights ([Table 1]).

Table 1 Physical characteristics of the three experimental groups

	SHAM	CTRL AF	RIVA AF	ANOVA p-Value
	n = 10	n = 10	n = 11
	Mean ± SD	Mean ± SD	Mean ± SD
Age (mo)	36.9 ± 17.2	39.9 ± 16.4	33.4 ± 13.7	0.64
Body weight (kg)	62.8 ± 11.8	66.5 ± 11.3	68.5 ± 11.6	0.54
Tibia length (cm)	25.5 ± 1.7	25.2 ± 2.9	24.1 ± 1.3	0.30
Organ weights (g)
Heart	320.1 ± 61.3	326.9 ± 72.2	309.4 ± 63.3	0.83
Lung	482.9 ± 76.0	465.2 ± 61.2	489.8 ± 72.9	0.72
Left kidney	77.8 ± 12.3	83.6 ± 15.8	75.1 ± 13.0	0.37
Right kidney	79.6 ± 12.6	83.6 ± 16.9	74.2 ± 13.5	0.35
Liver	903.7 ± 190.2	856.1 ± 176.0	873.2 ± 202.2	0.85
Ratios
Heart weight	12.5 ± 2.2	13.2 ± 3.5	12.8 ± 2.3	0.29
Tibia length
Heart weight	4.9 ± 0.6	4.9 ± 0.6	4.5 ± 0.5	0.27
Body weight

Note: Group comparisons were done by one-way ANOVA. SHAM n = 10, CTRL AF n = 10, RIVA AF n = 11 goats.

Twice-Daily Oral Rivaroxaban Administration Ensures Stable Rivaroxaban Plasma Levels in Goats

A prothrombin time (PT) prolongation of 20 to 50% was reached with plasma rivaroxaban concentrations between 0.025 and 0.061 µg/mL (Bayer AG, personal communication, December 2015). Plasma rivaroxaban levels were monitored at eight time points throughout the treatment period. At all time points, RIVA AF goats showed plasma rivaroxaban levels that were above the lower limit of the on-therapeutic range (0.025 µg/mL; [Supplementary Fig. S1D], available in the online version only).

Rivaroxaban Treatment Decreases Thrombin Generation Potential in Goats with AF

Rivaroxaban treatment resulted in a clear inhibition of clotting potential in RIVA AF from the moment prior to AF induction until 16 weeks of AF ([Table 2]). These results indicate that rivaroxaban effectively inhibited goat FXa, reducing the thrombin generation potential in goats with AF. Surprisingly, a slight decrease was observed in the CTRL AF group between the pre-AF and final time points. Despite this small decrease, thrombin generation parameters of CTRL AF and SHAM did not differ at the final time point.

Table 2 Thrombin generation assay parameters per group and different time points

	Pre-Riva	Pre-AF	Final
	Mean ± SD	Mean ± SD	Mean ± SD	p-Value
Lag time (min)
SHAM	–	–	5.2 ± 1.0	<0.001*,**
CTRL AF	–	3.9 ± 1.1	4.7 ± 1.2[a]
RIVA AF	4.2 ± 0.4	10.1 ± 2.3[b]	12.1 ± 3.4[b]
ETP (nM•min)
SHAM	–	–	942.3 ± 448.3	<0.001*,**
CTRL AF	–	841.9 ± 175.2	829.2 ± 121.4
RIVA AF	1,022.4 ± 258.9	466.7 ± 77.2[b]	454.9 ± 183.3[b]
Peak height (nM)
SHAM	–	–	193.5 ± 64.6	<0.001*,**
CTRL AF	–	196.2 ± 45.5	179.6 ± 25.7[a]
RIVA AF	248.9 ± 41.7	78.4 ± 15.4[b]	68.7 ± 33.0[b]
Start tail (min)
SHAM	–	–	22.4 ± 3.3	<0.001*,**
CTRL AF	–	20.2 ± 1.6	20.9 ± 1.7[a]
RIVA AF	20.5 ± 1.7	29.1 ± 3.5[b]	33.1 ± 5.5[b]

Note: Group comparisons for the final time point were done by Kruskall–Wallis test and Dunn's post hoc test for multiple comparisons (SHAM n = 10, CTRL AF n = 10, RIVA AF n = 11 goats): lag time * = RIVA AF versus SHAM (p < 0.001) and ** =  versus CTRL AF (p < 0.001), ETP * = RIVA AF versus SHAM (p < 0.001) and ** = versus CTRL AF (p = 0.002), peak height * = RIVA AF versus SHAM (p < 0.001) and ** = versus CTRL AF (p = 0.001), start tail * = RIVA AF versus SHAM (p = 0.003) and ** = versus CTRL AF (p < 0.001).

^a Paired samples t tests CTRL AF; pre-AF versus Final (n = 9): lag time p = 0.01, ETP p = 0.91 peak height p = 0.04, start tail p = 0.02.

^b Friedman test RIVA AF; pre-Riva versus pre-AF and versus Final (n = 11): lag time p < 0.001, ETP p < 0.001, peak height p < 0.001, start tail p < 0.001. For blood sampling timepoints, see [Fig. 1].

AF Induces Hemodynamic Changes which Are Not Altered by Rivaroxaban Treatment

The impact of AF and rivaroxaban treatment on goat hemodynamic function was assessed during the final experiment. In the CTRL AF and RIVA AF groups, pressures were measured during AF, while in SHAM animals, pressures were measured during SR. RIVA AF goats showed a significant reduction in cardiac output, LV systolic pressure, and maximal positive dP/dT (a measure of LV contractility) compared with SHAM ([Fig. 2F, I, K]). Similarly, a decrease in these parameters was also observed in CTRL AF goats, although this failed to reach statistical significance. Additionally, both CTRL AF and RIVA AF groups showed a significant increase in right atrial pressure compared with SHAM. The similarity in hemodynamic parameters between CTRL AF and RIVA AF suggests that rivaroxaban treatment does not affect AF-related hemodynamic changes in goats after 16 weeks.

Fig. 2 Representative ECG tracings, left ventricular (PLV) and right atrial (PRA) pressure curves per group (A–C). Hemodynamics, cardiac pressures per experimental group (D, F–L), and schematic representation of pressure-tip catheters used during the final experiment (E). Cardiac output, right atrial pressure, and aortic pressure group comparisons were done by one-way ANOVA and Tukey's post hoc test for multiple comparisons. Left ventricular pressures group comparisons were done by Kruskal–Wallis test and Dunn's post hoc test for multiple comparisons (^* p < 0.05, ^** p < 0.01). SHAM n = 10, CTRL AF n = 10, RIVA AF n = 9 goats.

AF Stabilizes Independently of Rivaroxaban Treatment

To monitor AF progression, the stability of AF during the first 48 hours of AF induction and the sensitivity of persistent AF to be terminated by flecainide were evaluated. In the first 48 hours, we assessed the number of AF paroxysms that were required to maintain AF over a period of a 4-hour interval ([Fig. 3A–C]). With time, AF paroxysms were less likely to spontaneously terminate, reducing the number of terminations/4 hours. However, no difference in the rate of progression between CTRL AF and RIVA AF was observed.

Fig. 3 AF stability during paroxysmal (A–C) and persistent AF (D). The number of spontaneous terminations (cardioversions) of AF paroxysms was determined in intervals of 4 hours. Hence, fewer paroxysms occur once AF becomes more stable. AF stability of persistent AF was determined using Flecainide. The cardioversion experiment outcome was tested for significance using Fisher's exact test on a 2 × 2 contingency table.

Pharmacological cardioversion experiments were performed at 3, 5, and 10 weeks of AF. The success rate of pharmacological cardioversion did not differ between CTRL AF and RIVA AF, indicating that the progressive AF stabilization was not significantly affected by rivaroxaban treatment ([Fig. 3D]).

AF Complexity During the Final Experiment

Atrial electrophysiological parameters measured during AF in the atrial free walls were assessed by epicardial mapping. A more complex AF pattern consists of a higher number of simultaneously propagating wavefronts, leading to a more dissociated activation pattern. Except for the left atria fractionation index, which was significantly higher in RIVA AF as compared with CTRL AF, rivaroxaban treatment did not affect any other AF complexity parameter in left (LA) and right (RA) atria ([Table 3]).

Table 3 AF complexity parameters determined in the left atrium and the right atrium

Left atrium	CTRL AF	RIVA AF	p-Value
	Mean ± SD	Mean ± SD
Waves (n/cycle)	10.7 ± 2.2	10.9 ± 3.9	0.85
Breakthroughs (n/cycle)	5.0 ± 1.3	5.4 ± 2.5	0.68
Conduction velocity (cm/s)	63.0 ± 4.9	66.0 ± 6.9	0.29
AF cycle length (ms)	124.1 ± 14.2	128.4 ± 12.8	0.29
Max. dissociation (ms)	25.9 ± 4.7	26.8 ± 6.0	0.70
Fractionation index	1.8 ± 0.4	2.6 ± 0.9	0.04
Right atrium
Waves (n/cycle)	5.9 ± 2.3	6.8 ± 2.9	0.45
Breakthroughs (n/cycle)	2.4 ± 1.2	3.1 ± 1.7	0.34
Conduction velocity (cm/s)	67.9 ± 11.6	65.8 ± 6.6	0.63
AF cycle length (ms)	104.7 ± 10.9	115.6 ± 13.2	0.06
Max. dissociation (ms)	17.9 ± 3.7	19.6 ± 6.1	0.47
Fractionation index	1.5 ± 0.4	1.6 ± 0.5	0.54

Note: Left atrial AF cycle length data were analyzed using Mann–Whitney test. All other group comparisons were done using student's t-test. CTRL AF n = 10, RIVA AF n = 10 goats.

Rivaroxaban Prevents Atrial Myocyte Hypertrophy in Goats with AF

Histological analysis of the LA and RA showed that the overall degree of endomysial fibrosis was comparable in the three study groups at the final time point. Similarly, further subdivision for the epicardial and endocardial layers of each atrium revealed no differences in endomysial fibrosis ([Fig. 4A–E]). Interestingly, atrial myocyte size was significantly increased in CTRL AF (13.5 µm [95% CI: 12.9, 14.0]) compared with SHAM (12.5 µm [95% CI: 12.0, 13.0]). This AF-related atrial myocyte hypertrophy was fully prevented in RIVA AF (12.2 µm [95% CI: 11.7, 12.7]; [Fig. 5A]). Further subdivision confirmed this finding in the LA epicardium, LA endocardium, and RA endocardium ([Fig. 5C, D, F]), demonstrating a protective effect of rivaroxaban against AF-mediated atrial myocyte hypertrophy in both atria.

Fig. 4 Histological analysis of atrial endomysial fibrosis. Endomysial fibrosis, quantified as cell-to-cell distance between neighboring myocytes (A), specified per atrium and cardiac wall (epicardium and endocardium, B–E). Mixed model analysis estimated overall means per group; SHAM: 2.81 µm (95% CI: 2.65, 2.97), CTRL AF: 2.84 µm (95% CI: 2.68, 3.00), RIVA AF: 2.90 µm (95% CI: 2.75, 3.06). SHAM n = 10, CTRL AF n = 10, RIVA AF n = 11 goats.

Fig. 5 Histological analysis of atrial myocyte hypertrophy. Myocyte hypertrophy specified per atrium and cardiac wall (A–E). Mixed model analysis estimated overall means per group; SHAM: 12.51 µm (95% CI: 11.99, 13.03), CTRL AF: 13.47 µm (95% CI: 12.94, 13.99), RIVA AF: 12.23 µm (95% CI: 11.73, 12.73). Significant pairwise comparisons: SHAM versus CTRL AF (p = 0.01), CTRL AF versus RIVA AF (p = 0.001). The significance of pairwise comparisons per atrium and cardiac wall is indicated in the graph. SHAM n = 10, CTRL AF n = 10, RIVA AF n = 11 goats.

AF Induces Atrial Pro-Fibrotic and Pro-Hypertrophic Gene Expression

A total of 14 genes were selected as targets for atrial qPCR analysis based on their documented role in either structural or vascular remodeling, coagulation, or inflammation ([Supplemental Table S2], available in the online version only).

In accordance with the increased myocyte size found in our histological analysis, RT-qPCR revealed that 16 weeks of AF upregulated two well-known pro-hypertrophic genes, NPPA and NPPB, in both the LA ([Table 4]) and the RA ([Table 5]). The overexpression of NPPA and NPPB was not prevented by rivaroxaban, suggesting that FXa inhibition may not be sufficient to bring the gene expression of these markers back to baseline levels.

Table 4 RT-PCR gene expression levels of the left atrium, expressed as fold change relative to SHAM

	SHAM			CTRL AF			RIVA AF
Left atrium	Ref.	SD	n	Fold change	SD	n	Fold change	SD	n	p-Value	SHAM vs. CTRL AF	CTRL AF vs. RIVA AF
ACTA2	1	0.47	9	1.83	0.88	10	2.10	1.10	10	0.04	0.05	0.66
ANGPT2	1	0.81	10	0.90	0.36	10	0.81	0.39	10	0.78	–	–
CAV1	1	0.31	10	1.61	0.44	10	1.57	0.50	11	0.007	0.004	0.71
CCL2	1	0.70	10	0.85	0.45	10	0.30	0.23	10	0.006	0.72	0.01
COL1A1	1	0.70	10	3.00	2.03	10	2.60	1.13	10	0.002	0.002	1.00
F2R	1	0.44	10	0.98	0.44	10	1.19	0.45	11	0.67	–	–
F2RL1	1	1.19	9	1.57	1.33	10	0.88	0.58	10	0.24	–	–
F2RL2	1	0.79	10	0.44	0.53	10	0.72	0.42	10	0.05	0.02	0.05
F3	1	0.92	10	0.48	0.31	9	0.57	0.70	10	0.50	–	–
IL6	1	0.82	10	0.66	0.47	9	0.51	0.60	10	0.13	–	–
NPPA	1	0.48	9	4.32	2.52	10	4.85	2.44	11	0.002	0.005	0.61
NPPB	1	1.08	9	22.64	16.97	10	27.91	20.88	11	<0.001	0.001	0.71
VEGFA	1	0.40	10	1.02	0.41	10	1.15	0.47	11	0.71	–	–
VWF	1	0.80	9	1.75	1.11	10	2.34	1.71	11	0.10	–	–

Note: Kruskal–Wallis test with Dunn's multiple comparisons post hoc test for the left atrium.

Table 5 RT-PCR gene expression levels of the right atrium, expressed as fold change relative to SHAM

	SHAM			CTRL AF			RIVA AF
Right atrium	Ref.	SD	n	Fold change	SD	n	Fold change	SD	n	p-Value	SHAM vs. CTRL AF	CTRL AF vs. RIVA AF
ACTA2	1	0.66	10	1.51	1.01	10	2.24	1.23	11	0.02	0.20	0.12
ANGPT2	1	0.68	10	0.96	0.42	10	1.33	0.83	11	0.39	–	–
CAV1	1	0.46	10	2.12	0.67	10	1.80	0.78	11	0.005	0.001	0.32
CCL2	1	0.75	10	0.78	0.36	10	0.46	0.30	11	0.08	–	–
COL1A1	1	0.53	10	0.75	0.96	10	1.05	1.27	11	0.20	–	–
F2R	1	0.48	10	0.52	0.24	10	0.89	0.44	11	0.04	0.01	0.10
F2RL1	1	0.68	10	0.07	0.08	9	0.56	0.70	11	0.01	0.002	0.08
F2RL2	1	0.31	9	1.17	1.10	9	1.12	1.34	10	0.70	–	–
F3	1	1.34	10	0.72	0.55	10	0.88	1.42	11	0.39	–	–
IL6	1	0.53	10	0.75	0.96	10	1.05	1.27	11	0.20	–	–
NPPA	1	0.61	10	2.77	1.34	10	3.12	1.86	11	0.001	0.002	0.83
NPPB	1	1.10	9	8.19	5.74	10	9.03	9.36	11	0.001	<0.001	0.60
VEGFA	1	0.71	10	1.04	0.32	10	1.80	1.49	11	0.18	–	–
VWF	1	0.85	10	1.64	0.78	10	2.63	2.09	11	0.07	–	–

Note: Kruskal–Wallis test with Dunn's multiple comparisons post hoc test for the right atrium.

Interestingly, LA expression of the pro-fibrotic genes ACTA2 and COL1A1 was upregulated after 16 weeks of AF in CTRL AF without an additional rivaroxaban effect. This suggests the presence of pro-fibrotic molecular changes in the goat LA during AF.

Our analysis also shed light on the expression of a key pro-inflammatory mediator, CCL2, and on the potential anti-inflammatory properties of rivaroxaban. We found that, although CCL2 was not upregulated by AF, rivaroxaban treatment significantly downregulated its expression in the LA. A similar expression pattern was found in the RA, although not significant (p = 0.13).

Finally, the expression of PAR-1–4 encoding genes (F2R, F2RL1, F2RL2, and F2RL3) was assessed. Goat LA and RA expressed PAR-1, -2, and -3 mRNAs, while PAR-4 mRNA was not detected. Specifically, PAR1 appears to be the most abundantly expressed PAR isoform in the goat atria ([Supplementary Fig. S4], available in the online version only). In the LA, PAR3 gene expression was downregulated by AF. In contrast, RA PAR3 did not show significant changes, but instead, PAR1 and PAR2 were both significantly downregulated by AF. The effect of AF on PAR mRNA expression in the goat atria was not affected by rivaroxaban.

Ultimately, CAV1, a gene encoding for an intracellular protein involved in PAR internalization, was found to be significantly upregulated in both atria after 16 weeks of AF. This effect was not altered by rivaroxaban treatment.

Thrombin-Mediated PAR-1 Activation Upregulates the Expression of Hypertrophic Genes in Human iPSC-CM

To elucidate the mechanisms underlying the protective effect of decreased thrombin generation on AF-induced atrial myocyte hypertrophy, in vitro studies were conducted on human atrial-like iPSC-CMs.

Direct activation of PAR-1 in human iPSC-CMs by the agonist TRAP14 significantly upregulated the mRNA expression of the hypertrophic gene NPPA (± twofold) and induced a trend toward the upregulation of NPPB (± fourfold; [Fig. 6A, B]). Additionally, TRAP14 significantly increased the expression of the pro-inflammatory gene CCL2 (± threefold) compared with control ([Fig. 6C]). These findings suggest that PAR-1 activation plays a direct role in promoting pro-hypertrophic and pro-inflammatory responses in atrial cardiomyocytes.

Fig. 6 Effect of PAR1 activation by TRAP14 and thrombin on NPPA, NPPB, and CCL2 gene expression (A–C, n = 4–10) and BNP protein secretion in iPSC-CM (D, n = 3–7). iPSC-CM were stimulated with TRAP14 (100 μM) or human thrombin (8 pm) either in the presence or absence of SCH79797 (1 μM) or dabigatran (350 ng/mL). Gene expression was measured by RT-qPCR after 24 hours of incubation. Results are expressed as fold-change relative to control, with dots indicating separate experiments and error bars indicating SD. Statistical analysis was performed using the nonparametric Kruskal–Wallis test with Dunn's multiple comparison post hoc test (^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001). BNP, brain natriuretic peptide; CCL2, chemokine (C-C motif) ligand 2; NPPA, natriuretic peptide A; NPPB, natriuretic peptide B; SD, standard deviation.

Similarly, exposure of iPSC-CM to thrombin, the most potent PAR-1 activator, led to a significant increase of NPPA (± threefold), NPPB (± sevenfold), and CCL2 (± sixfold) mRNA levels, which was prevented by dabigatran, the direct thrombin inhibitor ([Fig. 6A–C]).

Notably, the effect of thrombin on NPPB and CCL2 upregulation was also fully inhibited by the direct PAR-1 antagonist SCH79797 (NPPB: ± 14-fold decrease, CCL2: ± sixfold decrease), which also appeared to mitigate the thrombin-mediated upregulation of NPPA (± threefold decrease; [Fig. 6A–C]).

Finally, the overexpression of NPPB was accompanied by a trend toward increased BNP protein levels in the conditioned media of iPSC-CMs exposed to TRAP14 (± threefold) and thrombin (± twofold). In line with the gene expression data, the effect of thrombin on BNP levels was fully prevented by PAR-1 inhibition (± twofold decrease; [Fig. 6D]).

Together, these observations indicate that in human atrial cardiomyocytes, thrombin induces pro-hypertrophic and pro-inflammatory gene expression through PAR-1 activation.

Discussion

This study demonstrates that the FXa inhibitor rivaroxaban decreases thrombin generation and prevents AF-related atrial myocyte hypertrophy in the goat model of persistent AF. Additionally, this study shows that, in human iPSC-derived atrial-like cardiomyocytes, thrombin triggers hypertrophic responses via activation of PAR-1. Together, these results indicate that in vivo, rivaroxaban protects against AF-related myocyte hypertrophy via inhibition of FXa-mediated thrombin generation and reduced PAR-1 activation.

AF Stabilization in Goats Occurs Independently of Rivaroxaban Treatment

AF-related electrical remodeling (shortening of atrial refractoriness) takes place during the first couple of days after AF onset, while structural remodeling, which contributes to the stabilization of AF, develops over the following weeks to months.[24] [32] Several studies demonstrated that the success rate of pharmacological cardioversion declines over time in AF.[33] [34] In line with these observations, we found a decline in the success rate of flecainide cardioversions in goats, indicating progressive early AF stabilization.

At present, limited data are available on the impact of DOACs on AF stabilization and progression. In a mouse model of burst pacing-induced AF, rivaroxaban led to shorter AF episodes compared with placebo. Nevertheless, neither differences in AF inducibility nor in the duration of the effective refractory period were found between the rivaroxaban and placebo groups.[35] In accordance with these observations, we found that AF inducibility and stability were not affected by rivaroxaban treatment, suggesting that in healthy goats, anticoagulation initiated 1 week prior to and continued during AF induction is not sufficient to prevent progressive AF stabilization.

Pro-Fibrotic Changes in the Goat Atria During Atrial Fibrillation

A prominent feature of atrial structural remodeling is endomysial fibrosis, an increase in fibrous tissue between cardiomyocytes. We previously described that endomysial fibrosis occurs in the outer millimeter of the atrial wall, leading to significant conduction disturbances during AF.[36] [37]

In this study, endomysial fibrosis was not increased after 16 weeks of AF maintenance. Additionally, no differences in endomysial fibrosis were found across the subdivided atrial regions (LA and RA, endo- and epicardial layer). It should be noted that histological analysis was performed in frozen tissue, whereas, in previous studies, plastic-embedded specimens were used. This methodological discrepancy may have reduced the sensitivity for detecting fibrosis. In addition to this technical limitation, it is possible that in this model, lone AF produced only a mild degree of fibrosis, as AF is induced in otherwise healthy animals without preexisting comorbidities or structural heart disease. The combination of these factors, the methodological constraints, and the modest remodeling response associated with lone AF, may therefore explain the absence of detectable fibrosis at the histological level. Nevertheless, COL1A1 and ACTA2 were both upregulated in the LA of the CTRL AF group compared with SHAM. This overexpression indicates that, despite the lack of histological differences in goat atrial fibrotic content, pro-fibrotic molecular processes may be activated in the goat LA during AF.

Previous studies on small animal models have shown that rivaroxaban can reduce cardiac fibrotic processes.[35] [38] [39] For example, rivaroxaban administration decreased atrial interstitial fibrosis in a murine model of pressure overload and significantly attenuated atrial fibrosis in rats with isoproterenol-induced AF.[35] [39] However, in the goat model, rivaroxaban did not show any additional effect on endomysial fibrosis in AF. The lack of a difference in atrial fibrotic content between CTRL AF and RIVA AF goats was consistent with the comparable degree of AF complexity observed in both groups.

Rivaroxaban Treatment Prevents AF-Induced Atrial Myocytes Hypertrophy in Goats

Histological analyses revealed that 16 weeks of AF significantly increased both LA and RA myocyte size and upregulated the expression of the pro-hypertrophic genes NPPA and NPPB in both atria. Importantly, we demonstrate that oral rivaroxaban treatment decreased systemic thrombin generation and prevented atrial myocyte hypertrophy induced by AF in the goat model. While previous studies have shown that rivaroxaban treatment protects against ventricular hypertrophy in small, non-AF animal models, our study is the first to highlight an anti-hypertrophic effect of rivaroxaban within the atria in a large animal model of persistent AF.[40] [41]

It is important to acknowledge that, in addition to FXa and thrombin signaling, other mechanisms may contribute to atrial hypertrophic responses during AF. For instance, elevated atrial pressure may lead to atrial stretch, potentially triggering hypertrophic pathways independent of FXa signaling. In our study, rivaroxaban treatment did not prevent the AF-mediated increase in right atrial pressure, which may have acted as an additional hypertrophic stimulus. This may also explain the absence of downregulation of NPPA and NPPB expression by rivaroxaban in the goat atria.

Atrial Fibrillation Complexity

Both atrial myocyte hypertrophy and fibrosis have been related to electrophysiological changes that contribute to the development of complex fibrillatory conduction patterns.[18] [37] [42]

Our observation that myocyte diameter was uniformly increased throughout the atrial wall after a prolonged period of persistent AF aligns with previous findings.[19] [32] [42] However, the prevention of myocyte hypertrophy in the RIVA AF group did not reduce AF complexity, as none of the AF complexity parameters were lower in the RIVA AF group compared with the CTRL AF group. One possible explanation for this could be the relatively small difference in atrial myocyte size (approximately 10%) between the RIVA AF and CTRL AF goats, and/or the persistence of electrical remodeling processes, which may also independently contribute to more complex propagation patterns.[43]

PAR Gene Expression in Goat Atria with AF

Altered PAR expression has been observed in several pathological conditions. PAR-1 and -2 are more commonly expressed in the hearts of patients with ischemic and idiopathic dilated heart failure, while PAR-2 is also upregulated in the blood of patients with atherosclerosis.[44] [45] However, the relationship between PAR expression and underlying pathological mechanisms remains unclear, potentially due to species-specific differences in the regulation of PAR mRNA levels.

In mice, cardiac-specific PAR2 overexpression has been linked to larger hearts and altered expression of pro-hypertrophic genes.[44] Similarly, Pawlinski et al showed hypertrophic effects associated with PAR-1 overexpression, and reported that PAR1 deficiency in mice resulted in a significant reduction in LV dilation and impaired LV function after myocardial infarction.[16]

In contrast, studies in heart failure patients by Friebel et al[46] found that reduced PAR-2 expression in endomyocardial biopsies was associated with increased inflammatory infiltration and myocardial fibrosis.

Our analysis demonstrated measurable expression of PAR1–3 mRNAs, with PAR-1 being the most highly expressed isoform in goat atria. In the LA of the CTRL AF group, PAR-3 expression was significantly downregulated, while in the RA, PAR-1 and PAR-2 expression levels were reduced compared with SHAM. Interestingly, we also observed upregulation of CAV1, a gene encoding a protein involved in PAR internalization, in both the LA and RA, suggesting enhanced PAR internalization and/or recycling during AF.

PAR-1 Activation by Thrombin Induces Pro-Hypertrophic Responses in Human Atrial-Like iPSC-Derived Cardiomyocytes

In the goat model of AF, rivaroxaban inhibits FXa, leading to reduced systemic thrombin generation and attenuation of atrial myocyte hypertrophy.

To explore the mechanism by which thrombin and PAR signaling contribute to hypertrophic growth in atrial myocytes, we stimulated human atrial-like iPSC-CMs with thrombin or FXa ([Supplementary Figs. S2] and [S3], available in the online version only), with and without the selective PAR-1 antagonist SCH79797.

Although FXa can activate PARs and elicits pro-inflammatory responses in cardiac fibroblasts and other cardiac cell types, our in vitro experiments demonstrate that the hypertrophic signaling observed in human atrial-like iPSC-CMs was primarily driven by thrombin via PAR-1.[12] [47] Specifically, thrombin stimulation led to increased expression of NPPA and NPPB, which was significantly reduced by SCH79797, indicating that thrombin induces hypertrophic responses through PAR-1 activation. Furthermore, direct stimulation of PAR-1 using its selective agonist TRAP14 also significantly upregulated NPPA and NPPB expression, reinforcing the central role of PAR-1 in atrial cardiomyocyte hypertrophy.

To date, this study is the first to describe a direct pro-hypertrophic role of thrombin via PAR-1 in human atrial-like iPSC-derived cardiomyocytes, an effect previously observed only in rat atrial muscle preparations and neonatal rat ventricular cardiomyocytes.[48] [49]

Consistent with our findings, earlier studies on small animal models have shown that inhibition of PAR-1 activation by SCH79797 reduces cardiomyocyte hypertrophy in vivo, further supporting the pro-hypertrophic role of PAR-1 signaling.[17]

Importantly, our work extends these observations to a large animal model, offering a significant advance in translational relevance. Unlike small animal models of acute, short-term AF, the goat model of persistent AF closely mimics the clinical progression of the disease, as AF induction leads to a gradual increase in episode duration until AF becomes persistent and can be sustained for several months.[24] This unique feature enables detailed investigation of the long-term effects of anticoagulation therapy on atrial structural remodeling processes, which develop over days to months of sustained AF. In this context, our study is the first to demonstrate that sustained reduction of systemic thrombin generation exerts a protective effect against atrial myocyte hypertrophy in a large animal model with 16 weeks of AF.

Based on the identified hypertrophic effect of thrombin and PAR-1 signaling on human atrial-like iPSC-CMs, our findings indicate that in the goat model, rivaroxaban treatment effectively protects against AF-related myocyte hypertrophy by reducing FXa-mediated thrombin generation and decreasing PAR-1 activation.

Effect of Direct Oral Anticoagulants on Atrial Inflammatory Processes

This study also offers insights into the potential anti-inflammatory effects of DOACs. In human iPSC-CM, dabigatran effectively prevented thrombin-induced upregulation of the pro-inflammatory marker CCL2, a key regulator of inflammatory processes that encodes monocyte chemoattractant protein-1 (MCP1). Similarly, we and others have previously shown that rivaroxaban inhibits FXa-induced overexpression of CCL2 and IL6 in human primary cardiac fibroblasts.[12] [50] In the goat study, rivaroxaban downregulated CCL2 expression in the LA and showed a similar trend in the RA. As also supported by several mouse studies, our findings identify an important link between systemic inhibition of FXa and thrombin and reduced pro-inflammatory signaling in the heart.[35] [51]

Translational Outlook

This study describes, for the first time in a large animal model, the experimental evidence showing that systemic inhibition of FXa and thrombin during AF protects the heart from PAR-induced structural changes, like atrial myocyte hypertrophy, which plays a key role in the pathogenesis of AF. This study confirms the role of activated coagulation factors and PARs signaling in the pathophysiological mechanisms underlying AF-related structural remodeling, highlighting the potential of DOACs in preventing AF substrate development.

Although rivaroxaban exerts a protective effect against AF-induced atrial myocyte hypertrophy, our study shows that it does not directly influence the complexity or stability of persistent AF in goats. This finding underscores the multifactorial nature of the substrate sustaining long-lasting AF, in which several mechanisms, including electrical and contractile remodeling, can maintain AF even when myocyte hypertrophy is attenuated.

It is also important to emphasize that this study was performed in a goat model of lone AF, characterized by the absence of comorbidities or structural heart disease. This experimental context may account for the relatively modest increase in atrial myocyte size and structural remodeling observed in goats with AF. Accordingly, the protective effect of rivaroxaban in attenuating AF-induced myocyte hypertrophy resulted in a relatively small difference in myocyte size (approximately 10%) between groups, which may also explain the absence of a detectable impact of rivaroxaban on AF complexity.

In this context, it can be speculated that in patients with AF, who typically exhibit a more pronounced degree of atrial cardiomyopathy due to coexisting comorbidities and risk factors, the protective effects of rivaroxaban on atrial structural remodeling may become more evident, potentially leading to a less stable and less complex AF substrate.

Given that DOACs are the standard of care for stroke prevention in AF patients, their anti-inflammatory and anti-hypertrophic properties observed in this study may help prevent AF substrate development beyond their established role in stroke prevention. Randomized clinical trials comparing DOACs with placebo in at-risk patients are warranted to determine whether DOACs can reduce the incidence or delay the progression of AF.

Limitations

Four months of AF did not increase coagulation potential in goats, which contrasts with clinical studies that describe a hypercoagulable state in AF patients. In this study, coagulation assessment is only based on TG analysis, which might not be the most sensitive method to pick up changes in the goat coagulation, but direct immunoassays for quantifying thrombin, like F1 + 2, or thrombin-antithrombin complex, could not be used due to a lack of species cross-reactivity (data not shown). An alternative explanation can be that AF alone and/or AF duration might not be sufficient to trigger a systemic pro-coagulant response in goats in the absence of comorbidities like heart failure, diabetes, or vascular disease. In fact, AF patients without comorbidities typically show only limited activation of the coagulation system and little or no increase in stroke risk.[52] [53]

Furthermore, while we assessed the effect of AF on systemic coagulation, we did not examine the levels of specific coagulation factors in plasma or tissues due to known species differences that limit the use of commercial assays.

Human iPSC-CM were used as a cellular model for mechanistic studies. Despite iPSC-CM showing differentiation toward atrial-like cardiomyocytes at the gene expression level, sarcomere formation, and spontaneous beating ([Supplementary Figs. S5–S7], available in the online version only), when compared with adult cardiomyocytes, they show a more immature phenotype. Moreover, the complex and time-demanding iPSC differentiation process restricted the number of assays we could perform, limiting the power of our analysis.

Conclusion

In an established goat model of AF, prolonged oral treatment with the direct FXa inhibitor rivaroxaban provided a sustained reduction of systemic thrombin generation, resulting in diminished cardiomyocyte hypertrophy, a key element in the pathogenesis of AF. In human atrial-like iPSC-derived cardiomyocytes, thrombin triggered hypertrophic responses via activation of PAR-1. Taken together, these data indicate that in vivo, rivaroxaban protects against AF-related myocyte hypertrophy likely via inhibition of FXa-mediated thrombin generation and reduced PAR-1 activation.

Consistent with previous findings, this study further corroborates the role of activated coagulation factors and PAR signaling in AF-related structural remodeling processes and highlights the potential of DOACs in preventing AF substrate development.[11] The consequences for patients on long-term treatment with FXa-inhibiting DOACs merit further investigation.

Conflict of Interest

U.S. received consultancy fees or honoraria from Università della Svizzera Italiana (USI, Switzerland), Roche Diagnostics (Switzerland), EP Solutions Inc. (Switzerland), Johnson & Johnson Medical Limited (United Kingdom), and Bayer Healthcare (Germany). He received research grants from Roche and EP Solutions. Bayer Healthcare (Germany) supported the pharmacokinetic study of this investigation. He is co-founder and shareholder of YourRhythmics BV, a spin-off company of the University of Maastricht. H.t.C received consultancy fees from Bayer, Pfizer, Leo, Portola/Alexion, AstraZeneca, and received research grants from Bayer, unrelated to the present work. He is a consultant for Alveron and Novostia and a shareholder of CoagulationProfile, a spin-off company of Maastricht University.

Note

Portions of this manuscript are based on the doctoral dissertation of Scaf and D'Alessandro.[23]

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Publication History

Received: 16 September 2025

Accepted after revision: 02 December 2025

Accepted Manuscript online:
08 January 2026

Article published online:
19 January 2026