Primary cilia suppress the fibrotic activity of atrial fibroblasts from patients with atrial fibrillation in vitro.

Atrial fibrosis serves as an arrhythmogenic substrate in atrial fibrillation (AF) and contributes to AF persistence. Treating atrial fibrosis is challenging because atrial fibroblast activity is multifactorial. We hypothesized that the primary cilium regulates the profibrotic response of AF atrial fibroblasts, and explored therapeutic potentials of targeting primary cilia to treat fibrosis in AF. We included 25 patients without AF (non-AF) and 26 persistent AF patients (AF). Immunohistochemistry using a subset of the patients (non-AF: n = 10, AF: n = 10) showed less ciliated fibroblasts in AF versus non-AF. Acetylated α-tubulin protein levels were decreased in AF, while the gene expressions of AURKA and NEDD9 were highly increased in AF patients' left atrium. Loss of primary cilia in human atrial fibroblasts through IFT88 knockdown enhanced expression of ECM genes, including FN1 and COL1A1. Remarkably, restoration or elongation of primary cilia by an AURKA selective inhibitor or lithium chloride, respectively, prevented the increased expression of ECM genes induced by different profibrotic cytokines in atrial fibroblasts of AF patients. Our data reveal a novel mechanism underlying fibrotic substrate formation via primary cilia loss in AF atrial fibroblasts and suggest a therapeutic potential for abrogating atrial fibrosis by restoring primary cilia.

MicroRNAs in atrial fibrillation target genes in structural remodelling.

We aim to elucidate how miRNAs regulate the mRNA signature of atrial fibrillation (AF), to gain mechanistic insight and identify candidate targets for future therapies. We present combined miRNA-mRNA sequencing using atrial tissues of patient without AF (n = 22), with paroxysmal AF (n = 22) and with persistent AF (n = 20). mRNA sequencing previously uncovered upregulated epithelial to mesenchymal transition, endothelial cell proliferation and extracellular matrix remodelling involving glycoproteins and proteoglycans in AF. MiRNA co-sequencing discovered miRNAs regulating the mRNA expression changes. Key downregulated miRNAs included miR-135b-5p, miR-138-5p, miR-200a-3p, miR-200b-3p and miR-31-5p and key upregulated miRNAs were miR-144-3p, miR-15b-3p, miR-182-5p miR-18b-5p, miR-4306 and miR-206. MiRNA expression levels were negatively correlated with the expression levels of a multitude of predicted target genes. Downregulated miRNAs associated with increased gene expression are involved in upregulated epithelial and endothelial cell migration and glycosaminoglycan biosynthesis. In vitro inhibition of miR-135b-5p and miR-138-5p validated an effect of miRNAs on multiple predicted targets. Altogether, the discovered miRNAs may be explored in further functional studies as potential targets for anti-fibrotic therapies in AF.

Atrial epicardial adipose tissue abundantly secretes myeloperoxidase and activates atrial fibroblasts in patients with atrial fibrillation.

BACKGROUND: Epicardial adipose tissue (EAT) secretome induces fibrosis. Fibrosis, primarily extracellular matrix (ECM) produced by fibroblasts, creates a substrate for atrial fibrillation (AF). Whether the EAT secretome from patients with AF activates human atrial fibroblasts and through which components, remains unexplored. RESEARCH AIMS: (a) To investigate if the EAT secretome from patients with versus without AF increases ECM production in atrial fibroblasts. (b) To identify profibrotic proteins and processes in the EAT secretome and EAT from patients with, who will develop (future onset), and without AF. METHODS: Atrial EAT was obtainded during thoracoscopic ablation (AF, n = 20), or open-heart surgery (future onset and non-AF, n = 35). ECM gene expression of human atrial fibroblasts exposed to the EAT secretome and the proteomes of EAT secretome and EAT were assessed in patients with and without AF. Myeloperoxidase and neutrophil extracellular traps (NETs) were assessed immunohistochemically in patients with paroxysmal, persistent, future onset, and those who remain free of AF (non-AF). RESULTS: The expression of COL1A1 and FN1 in fibroblasts exposed to secretome from patients with AF was 3.7 and 4.7 times higher than in patients without AF (p < 0.05). Myeloperoxidase was the most increased protein in the EAT secretome and EAT from patients with versus without AF (FC 18.07 and 21.57, p < 0.005), as was the gene-set neutrophil degranulation. Immunohistochemically, myeloperoxidase was highest in persistent (FC 13.3, p < 0.0001) and increased in future onset AF (FC 2.4, p = 0.02) versus non-AF. Myeloperoxidase aggregated subepicardially and around fibrofatty infiltrates. NETs were increased in patients with persistent versus non-AF (p = 0.03). CONCLUSION: In AF, the EAT secretome induces ECM gene expression in atrial fibroblasts and contains abundant myeloperoxidase. EAT myeloperoxidase was increased prior to AF onset, and both myeloperoxidase and NETs were highest in persistent AF, highlighting the role of EAT neutrophils in the pathophysiology of AF.

Women Have More Recurrences of Atrial Fibrillation than Men after Thoracoscopic Ablation and Suffer More from Established Risk Factors.

INTRODUCTION: Atrial fibrillation (AF) is more prevalent in men than in women. However, women with AF are more symptomatic, have a worse quality of life, a higher stroke risk and may therefore benefit most from ablation. In this study we aim to identify the risk of recurrent AF after thoracoscopic ablation, and assess the differential impact of the risk factors for recurrence between women and men. METHOD: This is a single center cohort study, including patients undergoing thoracoscopic ablation for advanced AF between 2008 and 2019. All patients were clinically followed up for two years with quarterly 24 h Holter monitoring and ECGs for the detection of recurrent AF. Left atrial appendage (LAA) tissue was collected for collagen analysis. RESULTS: We included 571 patients, of whom 143 (25%) were women. Women were older than men (63 ± 8.3 y vs. 59 ± 8.5, p < 0.001), but had fewer cardiovascular risk factors, myocardial infarctions (1.4% vs. 6.5%, p = 0.03) and, in particular, vascular disease (7.0% vs. 16.1%, p = 0.01). Women suffered more from AF recurrence, driven by more atrial tachycardias, and sex was an independent risk factor for recurrence (HR1.41 [1.04-1.91], p = 0.028]). The presence of vascular disease was associated with an increased risk for AF recurrence in women, but not in men. In LAA histology, women had more collagen than men, as had patients with persistent compared to paroxysmal AF. CONCLUSION: Women had 15% more recurrences, driven by more atrial tachycardias, which may be explained by a more fibrotic atrial substrate. What's new? Women undergoing thoracoscopic AF ablation have a higher risk of recurrent AF, driven by more atrial tachycardias. Among patients with left atrial enlargement or persistent AF, women have worse outcomes than men. Vascular disease was a risk factor for recurrence in women, but not in men. In a histopathologic analysis of the left atrial appendage, women had more collagen than men, as had patients with persistent compared to paroxysmal AF.

Differential Sodium Current Remodelling Identifies Distinct Cellular Proarrhythmic Mechanisms in Paroxysmal vs Persistent Atrial Fibrillation.

BACKGROUND: The cellular mechanisms underlying progression from paroxysmal to persistent atrial fibrillation (AF) are not fully understood, but alterations in (late) sodium current (INa) have been proposed. Human studies investigating electrophysiological changes at the paroxysmal stage of AF are sparse, with the majority employing right atrial appendage cardiomyocytes (CMs). We here investigated action potential (AP) characteristics and (late) INa remodelling in left atrial appendage CMs (LAA-CMs) from patients with paroxysmal and persistent AF and patients in sinus rhythm (SR), as well as the potential contribution of the "neuronal" sodium channel SCN10A/NaV1.8. METHODS: Peak INa, late INa and AP properties were investigated through patch-clamp analysis on single LAA-CMs, whereas quantitative polymerase chain reaction was used to assess SCN5A/SCN10A expression levels in LAA tissue. RESULTS: In paroxysmal and persistent AF LAA-CMs, AP duration was shorter than in SR LAA-CMs. Compared with SR, peak INa and SCN5A expression were significantly decreased in paroxysmal AF, whereas they were restored to SR levels in persistent AF. Conversely, although late INa was unchanged in paroxysmal AF compared with SR, it was significantly increased in persistent AF. Peak or late Nav1.8-based INa was not detected in persistent AF LAA-CMs. Similarly, expression of SCN10A was not observed in LAAs at any stage. CONCLUSIONS: Our findings demonstrate differences in (late) INa remodeling in LAA-CMs from patients with paroxysmal vs persistent AF, indicating distinct cellular proarrhythmic mechanisms in different AF forms. These observations are of particular relevance when considering potential pharmacologic approaches targeting (late) INa in AF.

A failed catheter ablation of atrial fibrillation is associated with more advanced remodeling and reduced efficacy of further thoracoscopic ablation.

INTRODUCTION AND OBJECTIVES: Recent observations suggest that patients with a previous failed catheter ablation have an increased risk of atrial fibrillation (AF) recurrence after subsequent thoracoscopic AF ablation. We assessed the risk of AF recurrence in patients with a previous failed catheter ablation undergoing thoracoscopic ablation. METHODS: We included patients from 3 medical centers. To correct for potential heterogeneity, we performed propensity matching to compare AF freedom (freedom from any atrial tachyarrhythmia> 30 s during 1-year follow-up). Left atrial appendage tissue was analyzed for collagen distribution. RESULTS: A total of 705 patients were included, and 183 had a previous failed catheter ablation. These patients had fewer risk factors for AF recurrence than ablation naïve controls: smaller indexed left atrial volume (40.9± 12.5 vs 43.0±12.5 mL/m2, P=.048), less congestive heart failure (1.5% vs 8.9%, P=.001), and less persistent AF (52.2% vs 60.3%, P=.067). However, AF history duration was longer in patients with a previous failed catheter ablation (6.5 [4-10.5] vs 4 [2-8] years; P<.001). In propensity matched analysis, patients with a failed catheter ablation were at a 68% higher AF recurrence risk (OR, 1.68; 95%CI, 1.20-2.15; P=.034). AF freedom was 61.1% in patients with a previous failed catheter ablation vs 72.5% in ablation naïve matched controls. On histology of the left atrial appendage (n=198), patients with a failed catheter ablation had a higher density of collagen fibers. CONCLUSIONS: Patients with a prior failed catheter ablation had fewer risk factors for AF recurrence but more frequently had AF recurrence after thoracoscopic AF ablation than ablation naïve patients. This may in part be explained by more progressed, subclinical, atrial fibrosis formation.

Circulating adipose tissue proteins involved in atrial fibrillation: An explorative scoping review.

Obesity increases the risk of atrial fibrillation (AF), potentially through proteins secreted by adipose tissue (AT) that affect atrial electrical and structural remodeling. We aim to give a comprehensive overview of circulating AT proteins involved in inflammation and fibrosis, that are associated with prevalent AF (paroxysmal or persistent) and the risk on developing new-onset AF. These include adipokines, defined as proteins enriched in AT as adiponectin, but also proteins less specific to AT. We systematically performed an explorative search for studies reporting associations between proteins secreted from cells residing in the AT and AF, and additionally assessed the effect of obesity on these proteins by a secondary search. The AT proteins involved in inflammation were mostly increased in patients with prevalent and new-onset AF, and with obesity, while the AT enriched adipokines were mostly not associated with AF. This review provides insight into circulating adipose tissue proteins involved in AF substrate formation.

Personas for Better Targeted eHealth Technologies: User-Centered Design Approach.

BACKGROUND: The full potential of eHealth technologies to support self-management and disease management for patients with chronic diseases is not being reached. A possible explanation for these lacking results is that during the development process, insufficient attention is paid to the needs, wishes, and context of the prospective end users. To overcome such issues, the user-centered design practice of creating personas is widely accepted to ensure the fit between a technology and the target group or end users throughout all phases of development. OBJECTIVE: In this study, we integrate several approaches to persona development into the Persona Approach Twente to attain a more holistic and structured approach that aligns with the iterative process of eHealth development. METHODS: In 3 steps, a secondary analysis was carried out on different parts of the data set using the Partitioning Around Medoids clustering method. First, we used health-related electronic patient record data only. Second, we added person-related data that were gathered through interviews and questionnaires. Third, we added log data. RESULTS: In the first step, 2 clusters were found, with average silhouette widths of 0.12 and 0.27. In the second step, again 2 clusters were found, with average silhouette widths of 0.08 and 0.12. In the third step, 3 clusters were identified, with average silhouette widths of 0.09, 0.12, and 0.04. CONCLUSIONS: The Persona Approach Twente is applicable for mixed types of data and allows alignment of this user-centered design method to the iterative approach of eHealth development. A variety of characteristics can be used that stretches beyond (standardized) medical and demographic measurements. Challenges lie in data quality and fitness for (quantitative) clustering.

Thoracoscopic surgical atrial fibrillation ablation in patients with an extremely enlarged left atrium.

PURPOSE: Efficacy of pulmonary vein isolation (PVI) for atrial fibrillation (AF) decreases as left atrial (LA) volume increases. However, surgical AF ablation with unknown efficacy is being performed in patients with a giant LA (GLA). We determined efficacy of thoracoscopic AF ablation in patients with compared to without a GLA. METHODS: Patients underwent thoracoscopic PVI with additional left atrial ablations lines (in persistent AF) and were prospectively followed up. GLA was defined as LA volume index (LAVI) ≥ 50 ml/m2. Follow-up was performed with ECGs and 24-h Holters every 3 months. After a 3-month blanking period, all antiarrhythmic drugs were discontinued. The primary outcome was freedom of any atrial tachyarrhythmia ≥ 30 s during 2 years of follow-up. RESULTS: At baseline, 68 (15.4%) patients had a GLA (LAVI: 56.7 [52.4-62.8] ml/m2), while 374 (84.6%) had a smaller LA (LAVI: 34.8 [29.2-41.3] ml/m2). GLA patients were older (61.9 ± 6.9 vs 59.4 ± 8.8 years, p = 0.02), more often diagnosed with persistent AF (76.5% vs 58.6%, p = 0.008). Sex was equally distributed (with approximately 25% females). GLA patients had more recurrences compared to non-GLA patients at 2-year follow-up (42.6% vs 57.2%, log rank p = 0.02). Freedom of AF was 69.0% in non-GLA paroxysmal AF patients compared to 43.8-49.3% in a combined group of GLA and/or persistent AF patients(log rank p < 0.001). Furthermore, freedom was 62.4% in non-GLA male patients, compared to 43.8-47.4 in a combined group of GLA and/or female sex(log rank p = 0.02). CONCLUSION: Thoracoscopic AF ablation is an effective therapy in a substantial part of GLA patients. Thoracoscopic AF ablation may serve as a last resort treatment option in these patients.

Epicardial and endothelial cell activation concurs with extracellular matrix remodeling in atrial fibrillation.

BACKGROUND: Improved understanding of the interconnectedness of structural remodeling processes in atrial fibrillation (AF) in patients could identify targets for future therapies. METHODS: We present transcriptome sequencing of atrial tissues of patients without AF, with paroxysmal AF, and persistent AF (total n = 64). RNA expression levels were validated in the same and an independent cohort with qPCR. Biological processes were assessed with histological and immunohistochemical analyses. RESULTS: In AF patients, epicardial cell gene expression decreased, contrasting with an upregulation of epithelial-to-mesenchymal transition (EMT) and mesenchymal cell gene expression. Immunohistochemistry demonstrated thickening of the epicardium and an increased proportion of (myo)fibroblast-like cells in the myocardium, supporting enhanced EMT in AF. We furthermore report an upregulation of endothelial cell proliferation, angiogenesis, and endothelial signaling. EMT and endothelial cell proliferation concurred with increased interstitial (myo)fibroblast-like cells and extracellular matrix gene expression including enhanced tenascin-C, thrombospondins, biglycan, and versican. Morphological analyses discovered increased and redistributed glycosaminoglycans and collagens in the atria of AF patients. Signaling pathways, including cell-matrix interactions, PI3K-AKT, and Notch signaling that could regulate mesenchymal cell activation, were upregulated. CONCLUSION: Our results suggest that EMT and endothelial cell proliferation work in concert and characterize the (myo)fibroblast recruitment and ECM remodeling of AF. These processes could guide future research toward the discovery of targets for AF therapy.

Extracellular matrix remodeling precedes atrial fibrillation: Results of the PREDICT-AF trial.

BACKGROUND: To which extent atrial remodeling occurs before atrial fibrillation (AF) is unknown. OBJECTIVE: The PREventive left atrial appenDage resection for the predICtion of fuTure Atrial Fibrillation (PREDICT-AF) study investigated such subclinical remodeling, which may be used for risk stratification and AF prevention. METHODS: Patients (N = 150) without a history of AF with a CHA2DS2-VASc score of ≥2 at an increased risk of developing AF were included. The left atrial appendage was excised and blood samples were collected during elective cardiothoracic surgery for biomarker discovery. Participants were followed for 2 years with Holter monitoring to determine any atrial tachyarrhythmia after a 50-day blanking period. RESULTS: Eighteen patients (12%) developed incident AF, which was associated with increased tissue gene expression of collagen I (COL1A1), collagen III (COL3A1), and collagen VIII (COL8A2), tenascin-C (TNC), thrombospondin-2 (THBS2), and biglycan (BGN). Furthermore, the fibroblast activating endothelin-1 (EDN1) and sodium voltage-gated channel ß subunit 2 (SCN2B) were associated with incident AF whereas the Kir2.1 channel (KCNJ2) tended to downregulate. The plasma levels of COL8A2 and TNC correlated with tissue expression and predicted incident AF. A gene panel including tissue KCNJ2, COL1A1, COL8A2, and EDN1 outperformed clinical prediction models in discriminating incident AF. CONCLUSION: The PREDICT-AF study demonstrates that atrial remodeling occurs long before incident AF and implies future potential for early patient identification and therapies to prevent AF (ClinicalTrials.gov identifier NCT03130985).

Neutrophil degranulation interconnects over-represented biological processes in atrial fibrillation.

Despite our expanding knowledge about the mechanism underlying atrial fibrillation (AF), the interplay between the biological events underlying AF remains incompletely understood. This study aimed to identify the functionally enriched gene-sets in AF and capture their interconnection via pivotal factors, that may drive or be driven by AF. Global abundance of the proteins in the left atrium of AF patients compared to control patients (n = 3/group), and the functionally enriched biological processes in AF were determined by mass-spectrometry and gene set enrichment analysis, respectively. The data were validated in an independent cohort (n = 19-20/group). In AF, the gene-sets of innate immune system, metabolic process, cellular component disassembly and ion homeostasis were up-regulated, while the gene-set of ciliogenesis was down-regulated. The innate immune system was over-represented by neutrophil degranulation, the components of which were extensively shared by other gene-sets altered in AF. In the independent cohort, an activated form of neutrophils was more present in the left atrium of AF patients with the increased gene expression of neutrophil granules. MYH10, required for ciliogenesis, was decreased in the atrial fibroblasts of AF patients. We report the increased neutrophil degranulation appears to play a pivotal role, and affects multiple biological processes altered in AF.

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

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.

Visualisation of the 'Optimal Cerebral Perfusion' Landscape in Severe Traumatic Brain Injury Patients.

OBJECTIVE: An 'optimal' cerebral perfusion pressure (CPPopt) can be defined as the point on the CPP scale corresponding to the greatest autoregulatory capacity. This can be established by examining the pressure reactivity index PRx-CPP relationship, which is approximately U-shaped but suffers from noise and missing data. In this paper, we present a method for plotting the whole PRx-CPP relationship curve against time in the form of a colour-coded map depicting the 'landscape' of that relationship extending back for several hours and to display this robustly at the bedside.This is a short version of a full paper recently published in Critical Care Medicine (2016) containing some new insights and details of a novel bedside implementation based on a presentation during Intracranial Pressure 2016 Symposium in Boston. METHODS: Recordings from routine monitoring of traumatic brain injury patients were processed using ICM+. Time-averaged means for arterial blood pressure, intracranial pressure, cerebral perfusion pressure (CPP) and pressure reactivity index (PRx) were calculated and stored with time resolution of 1 min. ICM+ functions have been extended to include not just an algorithm of automatic calculation of CPPopt but also the 'CPPopt landscape' chart. RESULTS: Examining the 'CPPopt landscape' allows the clinician to differentiate periods where the autoregulatory range is narrow and needs to be targeted from periods when the patient is generally haemodynamically stable, allowing for more relaxed CPP management. This information would not have been conveyed using the original visualisation approaches. CONCLUSIONS: We describe here a natural extension to the concept of autoregulatory assessment, providing the retrospective 'landscape' of the PRx-CPP relationship extending over the past several hours. We have incorporated such visualisation techniques online in ICM+. The proposed visualisation may facilitate clinical evaluation and use of autoregulation-guided therapy.

Enhanced Visualization of Optimal Cerebral Perfusion Pressure Over Time to Support Clinical Decision Making.

OBJECTIVE: Cerebrovascular reactivity can provide a continuously updated individualized target for management of cerebral perfusion pressure, termed optimal cerebral perfusion pressure. The objective of this project was to find a way of improving the optimal cerebral perfusion pressure methodology by introducing a new visualization method. DATA SOURCES: Four severe traumatic brain injury patients with intracranial pressure monitoring. DATA EXTRACTION: Data were collected and pre-processed using ICM+ software. DATA SYNTHESIS: Sequential optimal cerebral perfusion pressure curves were used to create a color-coded maps of autoregulation - cerebral perfusion pressure relationship evolution over time. CONCLUSIONS: The visualization method addresses some of the main drawbacks of the original methodology and might bring the potential for its clinical application closer.