Reduced left atrial cardiomyocyte PITX2 and elevated circulating BMP10 predict atrial fibrillation after ablation

Jasmeet S Reyat, Winnie Chua, Victor R Cardoso, Anika Witten, Peter M Kastner, S Nashitha Kabir, Moritz F Sinner, Robin Wesselink, Andrew P Holmes, Davor Pavlovic, Monika Stoll, Stefan Kääb, Georgios V Gkoutos, Joris R de Groot, Paulus Kirchhof, Larissa Fabritz, Jasmeet S Reyat, Winnie Chua, Victor R Cardoso, Anika Witten, Peter M Kastner, S Nashitha Kabir, Moritz F Sinner, Robin Wesselink, Andrew P Holmes, Davor Pavlovic, Monika Stoll, Stefan Kääb, Georgios V Gkoutos, Joris R de Groot, Paulus Kirchhof, Larissa Fabritz

Abstract

BACKGROUNDGenomic and experimental studies suggest a role for PITX2 in atrial fibrillation (AF). To assess if this association is relevant for recurrent AF in patients, we tested whether left atrial PITX2 affects recurrent AF after AF ablation.METHODSmRNA concentrations of PITX2 and its cardiac isoform, PITX2c, were quantified in left atrial appendages (LAAs) from patients undergoing thoracoscopic AF ablation, either in whole LAA tissue (n = 83) or in LAA cardiomyocytes (n = 52), and combined with clinical parameters to predict AF recurrence. Literature suggests that BMP10 is a PITX2-repressed, atrial-specific, secreted protein. BMP10 plasma concentrations were combined with 11 cardiovascular biomarkers and clinical parameters to predict recurrent AF after catheter ablation in 359 patients.RESULTSReduced concentrations of cardiomyocyte PITX2, but not whole LAA tissue PITX2, were associated with AF recurrence after thoracoscopic AF ablation (16% decreased recurrence per 2-(ΔΔCt) increase in PITX2). RNA sequencing, quantitative PCR, and Western blotting confirmed that BMP10 is one of the most PITX2-repressed atrial genes. Left atrial size (HR per mm increase [95% CI], 1.055 [1.028, 1.082]); nonparoxysmal AF (HR 1.672 [1.206, 2.318]), and elevated BMP10 (HR 1.339 [CI 1.159, 1.546] per quartile increase) were predictive of recurrent AF. BMP10 outperformed 11 other cardiovascular biomarkers in predicting recurrent AF.CONCLUSIONSReduced left atrial cardiomyocyte PITX2 and elevated plasma concentrations of the PITX2-repressed, secreted atrial protein BMP10 identify patients at risk of recurrent AF after ablation.TRIAL REGISTRATIONClinicalTrials.gov NCT01091389, NL50069.018.14, Dutch National Registry of Clinical Research Projects EK494-16.FUNDINGBritish Heart Foundation, European Union (H2020), Leducq Foundation.

Keywords: Arrhythmias; Cardiology.

Conflict of interest statement

Conflict of interest: MS, MFS, SK, LF, and PK received support from the European Union CATCH ME. JRDG reports grants from Atricure Inc., Boston Scientific, Abbott, and Medtronic; personal fees from Atricure Inc., Bayer, Daiichi Sankyo, Servier, Johnson & Johnson, Medtronic, and Novartis. PK and LF receive research support for basic, translational, and clinical research projects from the British Heart Foundation, Leducq Foundation, Medical Research Council (United Kingdom), and German Centre for Cardiovascular Research; and from several drug and device companies active in atrial fibrillation. PK has previously received honoraria from several such companies. PK and LF are listed as inventors on 2 patents held by the University of Birmingham (“Atrial fibrillation therapy,” WO 2015140571; “Markers for atrial fibrillation,” WO 2016012783).

Figures

Figure 1. Flow diagram of patients included…
Figure 1. Flow diagram of patients included in the study, and analysis plan.
Figure 2. Expression of PITX2 or PITX2c…
Figure 2. Expression of PITX2 or PITX2c in whole left atrial tissue does not predict recurrent atrial fibrillation.
Left atrial appendage samples were digested and assessed for levels of PITX2 (A; PITX2 median [Q1, Q3] 8.67 [3.90, 16.78]) and PITX2c (B; 0.47 [0.16, 1.20]) using qPCR. Results are expressed as an average normalized to 2 housekeeping genes (GAPDH and POLR2A) (n = 94). Expression levels of PITX2 (C; PITX2 No AF Rec 7.81 [3.96, 16.72]), Rec AF 11.28 [3.70, 16.96]; P = 0.704) and PITX2c (D; PITX2c No AF Rec 0.44 [0.18, 1.19], Rec AF 0.53 [0.16, 1.50]; P = 0.543) were stratified by clinical outcomes of having recurrent AF within 1 year after ablation surgery. AF Rec, patients with recurrent AF, n = 23; No AF Rec, patients without recurrent AF, n = 71. (E) Example biopsies of left atrial appendage tissue, highlighting tissue heterogeneity. Scale bar: 10 mm.
Figure 3. Assessing PITX2 levels in patient…
Figure 3. Assessing PITX2 levels in patient left atrial cardiomyocytes.
(A) Overview of tissue processing and PITX2 gene expression and analysis protocol. Nuclei were isolated from patient left atrial appendage samples, and cardiomyocytes (CM) were enriched using an anti-PCM1 antibody. Both PCM1-enriched (CM Nuclei) and -depleted (Non-CM Nuclei) fractions were harvested. (B) Quantity of nuclei was assessed by staining using DAPI and determined by flow cytometry. (C) The percentage of nuclei in either non-CM or CM fractions was calculated (Non-CM 76.09 [66.06, 90.22], CM 23.91 [9.78, 33.94]; n = 52). (D) Levels of PITX2 (Non-CM 0.48 [0.19, 0.85], CM 4.43 [2.49, 8.39]; P < 0.001; n = 52) and (E) vWF (Non-CM 15.88 [13.12, 19.92], CM 0.44 [0.28, 0.58]; P < 0.001; n = 8) in both Non-CM and CM fractions were measured using qPCR. The results are expressed as an average normalized to 2 housekeeping genes (GAPDH and POLR2A). Statistical significance was calculated by using Mann-Whitney U test.
Figure 4. Reduced expression of PITX2 in…
Figure 4. Reduced expression of PITX2 in patient left atrial cardiomyocytes predicts recurrent atrial fibrillation.
(A) Expression levels of PITX2 in patient nuclei. Samples were stratified by AF recurrence at 1 year follow-up after ablation (No AF Rec 5.58 [3.16, 8.80], AF Rec 3.32 [1.60, 6.25]; P < 0.082. AF Rec, patients with recurrent AF within 1 year after ablation, n = 16; No AF Rec, patients without recurrent AF within 1 year after ablation, n = 36. (B) Stratification of PITX2 mRNA concentrations from A into quartiles. The numbers of patients who experienced recurrent AF in the respective quartiles are shown.
Figure 5. Bmp10 expression is increased following…
Figure 5. Bmp10 expression is increased following reduction of Pitx2.
(A) RNA-Seq analysis of significantly upregulated genes in left atrial tissue from Pitx2c+/– mice (n = 3 mouse pairs). (B) Bmp10 mRNA expression levels in the left atrium (LA) and left ventricle (LV) of WT and Pitx2c+/– mice, assessed by qPCR using Gapdh as a housekeeping gene (WT LA 0.03 [0.01, 0.04], Pitx2c+/– LA 3.20 [2.86, 3.60], P = 0.002; WT LV 0.05 [0.01, 0.09], Pitx2c+/– 0.01 [0.01, 0.02], P = 0.060; n = 6). Statistical significance was calculated using Mann-Whitney U test. (C) Protein expression of Bmp10 in the left atrium and left ventricle of WT and Pixt2+/– mice as assessed by Western blotting using Gapdh as a loading control (WT LA 1.00 [1.00, 1.00], Pitx2c+/– LA 2.34 [1.43, 3.05], P < 0.059; WT LV 0.40 [0.22, 0.78], Pitx2c+/– 0.34 [0.16, 0.45], P < 0.462; n = 4). (D) BMP10 mRNA expression levels in human left atrial cardiomyocyte (CM) and non-cardiomyocyte (Non-CM) appendage samples assessed by qPCR using GAPDH as a housekeeping gene (Non-CM 0.00 [0.00, 0.00] CM 0.70 [0.45, 1.95]; P = 0.032, n = 8). Statistical significance was calculated using a 1-sample t test.
Figure 6. Increased BMP10 predicts recurrent atrial…
Figure 6. Increased BMP10 predicts recurrent atrial fibrillation in 359 patients after catheter ablation.
(A) By univariate analysis, BMP10 confers the highest relative risk for recurrent AF among 11 other common cardiovascular biomarkers after adjustment for age, sex, type of AF, and left atrial diameter. (B) BMP10 levels are significantly elevated in patients with recurrent AF. (C) In multivariate analysis, increased left atrial (LA) size, nonparoxysmal type of AF, and elevated BMP10 predict recurrent AF. (D) When patients were stratified into quartiles based on BMP10 concentration, the largest proportion of patients with recurrent AF were in the highest BMP10 quartile. The numbers of patients that experienced recurrent AF in the respective quartiles are shown. (E) Stratification of patients by BMP10 quartiles corresponds with their rhythm outcome up to 2 years follow-up, with the worst outcomes in patients in the highest quartile (Q4). ANG2, angiopoietin 2; CRP, high-sensitivity C-reactive protein; CA125, cancer antigen 125; ESM1, endothelial cell–specific molecule 1; FABP3, fatty acid binding protein 3; GDF15, growth differentiation factor 15; IGFBP7, insulin like growth factor binding protein 7; TnT, high-sensitivity cardiac troponin T.
Figure 7. Correlation of low left atrial…
Figure 7. Correlation of low left atrial cardiomyocyte PITX2 mRNA and elevated BMP10 protein concentrations with recurrent AF after ablation.
Our data show that left atrial cardiomyocyte PITX2 mRNA concentrations, measured in left atrial appendages excised after thoracoscopic AF ablation, are a strong predictor of recurrent AF after AF ablation. Based on molecular biology analyses, we postulate that PITX2 represses production of the left atrial protein BMP10 that is secreted into blood. Indeed, elevated concentrations of BMP10 in peripheral blood were found to predict recurrent AF after AF ablation. These data call for validation in independent cohorts.

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