Atrial Appendages Research Articles

Insights into type 2 diabetes mellitus in atrial fibrillation patients: a proteomics approach

Abstract Introduction Atrial fibrillation (AF) and Type 2 Diabetes Mellitus (T2DM) are two closely interconnected conditions, sharing a complex relationship within cardiac pathophysiology. Common risk factors such as obesity, hypertension, inflammation, and endothelial dysfunction contribute to their coexistence. T2DM is known to drive cardiac fibrosis and promote electrical and structural remodeling of the heart, creating a substrate conducive to AF onset, while AF in T2DM patients increases the risk of adverse cardiovascular outcomes, such as stroke. Understanding this complex relationship is pivotal for improving the therapeutic and preventive strategies for these patients. This study aims to examine the proteomic differences between AF patients with and without T2DM, providing novel insights into pathophysiological mechanisms and identifying novel therapeutic targets and biomarkers. Methods In total, 14 AF patients undergoing open-heart surgery, comprising of 6 T2DM individuals and 8 non-T2DM individuals have been included. The mean age of the T2DM patients was 69.5 ± 10 years, while the non-T2DM was 69.3 ± 8.2 and BMI was 29.2 ± 2.9 and 29.5 ± 3 respectively. Right atrial appendages (RAA) from these patients were obtained in an RNA stabilization solution during open-heart surgery and processed using the phenol-based method for protein extraction. The proteins were digested with trypsin to generate peptides, followed by an additional clean-up step using C18 pipette tips. The prepared peptide samples were analyzed by employing a label-free-based quantitative (LFQ) approach, using a C18 (75cmx75cm) column. Proteomic analysis was performed using a Nano liquid chromatography system coupled to tandem mass spectrometer (nano LC-MS/MS), followed by a comprehensive bioinformatics analysis, using appropriate software. Results A total of 1548 proteins were identified, of which 899 could be quantified using LFQ analysis. Applying strict significance criteria, including FDR <0.1 and Abundance ratio (AR)>2 or <0.5, we identified 100 differential expressed proteins, with 21 upregulated and 79 downregulated in T2DM (Fig.1). Most notably, DPP9 and ATP1A2 were overexpressed in hearts of AF patients with T2DM (AR=100), while INPPL1 and ARID5A were underexpressed (AR=0.01), thereby representing attractive therapeutic targets. In pathway enrichment analysis (Fig.2), the dysregulated proteins participate in noteworthy pathways, including Non-alcoholic fatty liver disease (NAFLD), Type II diabetes mellitus, and Insulin signaling pathway. Conclusion Given the increased morbidity associated with the coexistence of AF and T2DM, there is a pressing need for novel therapeutic strategies tailored to this patient population. While AF and T2DM share certain molecular characteristics in the heart, this study emphasizes the necessity for further evaluation and validation of the distinct proteomic signatures identified.Fig.1:Volcano PlotFig.2:Protein Enrichment Network

Investigating the role of an orphan nuclear receptor NR4A1 in atrial fibrillation

Abstract Introduction Atrial fibrosis is a major remodelling process in atrial fibrillation (AF). The lack of clinically effective drug targets, due to insufficient understanding of the mechanisms of cardiac fibrosis, hampers the treatment of AF. Orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) is linked to fibrotic disease in multiple organs, however, its role in cardiac fibrosis and AF is yet to be demonstrated. Recent transcriptomic profiling revealed that NR4A1 expression is downregulated in human atrial cardiac fibroblasts (ACFs) in the presence of persistent AF. However, its mechanistic role in atrial fibrogenesis and AF is unknown. Purpose To investigate the expression profile and function of NR4A1 in human ACFs and provide insights into the role of NR4A1 in the context of persistent AF. Methods Human ACFs were isolated (enzymatic digestion) from right and left atrial appendages of 24 patients with persistent AF and controls in sinus rhythm (SR), who had elective heart surgery. NR4A1 knockdown in ACFs was achieved using NR4A1-siRNA. Gene and protein expression were determined by qPCR and western blot respectively, while BrdU assay and scratch assay were used to evaluate cell proliferation and migration. Atrial fibroblasts subpopulations were identified by single-nucleus RNA-sequencing. Subcellular localisation of NR4A1 was assessed by immunostaining, analysed by ImageJ. Results Validation of RNA-sequencing data confirmed a reduction of NR4A1 gene expression in human ACFs in persistent AF by 25% (p=0.029, Fig. 1A) and the NR4A1 predominant abundance in one of three identified subclusters of human ACFs (NR4A1+NAMPT+). The siRNA-mediated knockdown of NR4A1 in ACFs suppressed gene and protein expression of important component of extracellular matrix collagen-1 (Fig. 1B and Fig. 1C) and fibronectin protein (but not mRNA). The NR4A1-deficient cells also had decreased alpha-smooth muscle actin (αSMA) protein and gene expression (Fig. 1D and Fig. 1E), reduced periostin gene expression in the absence of changes in its protein. Furthermore, the NR4A1-deficiency reduced proliferation (by 18%, p=0.005, Fig. 1F) and accelerated migration (p=0.019, Fig. 1G) of human ACFs. These effects were independent of TGF-β1 stimulation, unlike reported in rat neonatal cardiac and dermal fibroblasts. Despite decreased NR4A1 gene expression, patients with AF had doubled NR4A1 protein levels compared to SR-ACFs (p=0.049, Fig. 2A). This was associated with abnormal 1.8-fold increase in the receptor subcellular localisation (p=0.032, Fig. 2B and Fig. 2C), as opposed to the physiological nuclear localisation observed in ACFs in the absence of AF. Conclusion While under physiological conditions, NR4A1 potently activates profibrotic processes in human ACFs, persistent AF patients have increased but abnormally distributed NR4A1 protein in atrial fibroblasts, whose consequences are yet to be determined in order to develop new therapies for AF.Figure 1Figure 2

Integrating single-cell sequencing and genetic lineage tracing reveals endothelial plasticity during atrial remodeling

Abstract Background Atrial fibrillation (AF), as the most burdensome cardiac arrhythmia, could lead to the significant mortality and morbidity. Atrial fibrosis is a well-recognized pathophysiological factor of the development and progression of AF, which also hampers its treatment. However, the underlying mechanisms of this intricate process remain largely unknown. Purpose This study sought to reveal the cellular composition of AF substrate and investigate the vital contributors to atrial structural remodeling. Methods Left atrial appendages from three patients with AF undergoing surgical ablation and three individuals with sinus rhythm undergoing heart transplantation were subjected to 10X single-cell RNA sequencing (scRNA-seq). Another five public scRNA-seq or snRNA-seq datasets of potentially remodeled right or left appendages were retrieved across three publications. The vital contributors of atrial structural remodeling and their characteristics were identified by intercellular communication analysis and trajectory inference. Transverse aortic constriction (TAC) was used to induce atrial remodeling in Cdh5-CreERT2;RFP mice, followed by scRNA-seq of endothelial cells (ECs). Human primary atrial ECs and fibroblasts were isolated for cell-cell interaction experiments. ECs-specific mouse models of Transforming growth factor beta 1 (TGFB1) (knockout and overexpression) were generated. Electrophysiological and histology analyses were performed to determine the consequences of ECs-derived TGFB1 gain and loss of function in the atrium. Results The comprehensive analysis of different scRNA-seq datasets revealed that ECs played an important regulatory role in the homeostasis of atrial substrate and displayed remarkable mesenchymal activation during atrial remodeling, which was further confirmed by TAC mice. Immunofluorescence staining and high-content screening suggested that the complete transition from ECs to mesenchymal cells slightly contributed to atrial remodeling. However, TGFB1 secreted from mesenchymal activated ECs significantly promoted activation, proliferation, and collagen secretion of fibroblasts. We discovered that overexpression of TGFB1 in ECs of mice significantly increased atrial fibrosis, and thus prolonging AF duration. Besides, knockout of TGFB1 in ECs of mice underwent TAC significantly attenuated the atrial fibrosis and decreased the rate of AF. Conclusion Our work demonstrates that mesenchymal ECs-derived Tgfb1 plays an important role in atrial remodeling by regulating the fibroblast function. EC-specific interventions were suggested to facilitate the development of novel strategies for mitigating AF substrate remodeling.

Interleukin-6 levels in atrium and onset of spontaneous atrial fibrillation: is there a causal relationship

Abstract Background Cumulative evidence suggests that atrial interleukin-6 (IL-6) may play a significant role in the development of spontaneous postoperative atrial fibrillation (sPOAF) in coronary artery bypass grafting (CABG) patients. Our previous studies found that the IL-6 produced by the atrium is positively related to the development of sPOAF. However, the causal relationship between IL-6 and sPOAF is not established. Purpose This study aims to explore the potential causality between atrial IL-6 and spontaneous AF(sAF), alongside assessing the impact of IL-6 on the electrophysiological properties of atrial myocytes (AM). Methods To determine the causal link between IL-6 and sAF, mice were randomly divided into eight groups: seven receiving injections of varying doses of recombinant IL-6 protein groups (15ng/kg to180ng/kg) into the left atrial wall, and one control group (vehicle). Mice were monitored for sAF for 7 days using implanted telemetry device. At 48 hrs post-injection, the left atrial appendages of another set of mice were collected for analyzing levels of IL-6, Myeloperoxidase (MPO), Transforming Growth Factor-β1 (TGF-β1), and Tumor Necrosis Factor-α (TNF-α). To assess the effect of IL-6 on the electrophysiological characteristics of AM, AMs were isolated from the hearts of 12-week-old mice and divided into experimental and control groups for electrophysiological testing. The experimental group was perfused with extracellular solutions containing 100 ng/mL of IL-6 while the control group was perfused with standard extracellular solutions. The measurements focused on action potential (AP), transient outward potassium current (Ito), and inward rectifier potassium current (Ik1) after a 30-minute perfusion. Results As depicted in Fig. 1A, a dose-response relationship between IL-6 levels and sAF occurrence was observed, with incidence increasing from 0% at 15 ng/kg to 75% at 180 ng/kg (χ²= 16.691, P = 0.005). The injection of IL-6 also induced the production of MPO, TGF-β1, and TNF-α with a dose-dependent effects (Fig. 2). In comparison to control, the AP duration at 90% (APD90) and APD50 of repolarization of atrial myocytes were significantly shortened in IL-6 group (Fig. 1B). For voltages from -10mV to +70mV, atrial myocytes in the IL-6 group showed significantly lower Ito current density than those in the control (Fig. 1C). No significant alterations were observed in the impact of IL-6 on Ik1 current density in atrial myocytes (Fig.1D). Conclusion To the best of our knowledge, this is the first study that experimentally tested the causal relation between IL-6 and sAF. The injection of IL-6 could cause the onset of sAF and produce MPO, TGF-β1, and TNF-α with a significant dose-dependent effect. The effect of IL-6 on inducing sAF might be through its effect on reducing APD and Ito current density in mouse atrial myocytes. This suggests that inhibition of atrial IL-6 might be a potential novel sPOAF prevention strategy.

Ablation Strategies for Repeat Procedures in Atrial Fibrillation Recurrences Despite Durable Pulmonary Vein Isolation: The Prospective Randomized ASTRO AF Multicenter Trial.

Ablation strategies for patients with symptomatic atrial fibrillation and isolated pulmonary veins vary and their effects on arrhythmia recurrence remain unclear. A prospective randomized German multicenter trial sought to compare 2 ablation strategies in this patient cohort. Patients with atrial fibrillation despite durable pulmonary vein isolation were randomly assigned at 7 centers to undergo low-voltage area ablation using 3-dimensional mapping and irrigated radiofrequency current ablation (group A) or empirical left atrial appendage isolation (LAAI) using the cryoballoon followed by staged interventional left atrial appendage closure (group B). The primary end point was freedom from atrial tachyarrhythmias between 91 and 365 days after index ablation. The study was powered for superiority of LAAI compared with low-voltage area. Patients (40% women; mean age, 68.8±8 years) with paroxysmal (32%) or persistent atrial fibrillation (68%) were randomized to undergo low-voltage area ablation (n=79) or cryoballoon-guided LAAI (n=82). After a planned interim analysis, enrollment was halted for futility on January 10, 2023. In the LAAI group, 77 of 82 left atrial appendages were successfully isolated with subsequent left atrial appendage closure in 57 patients. Procedure-related complications occurred in 4 (5%) and 11 (13.5%) patients in group A and B, respectively (P=0.10). The median follow-up was 367 days (interquartile range, 359-378). The Kaplan-Meier point estimate for freedom from atrial tachyarrhythmias was 51.7% (CI, 40.9%-65.4%) for group A and 55.5% (CI, 44.4%-69.2%; P=0.8069) for group B. The current study did not detect superiority of cryoballoon-guided LAAI over low-voltage area ablation in patients with atrial fibrillation despite durable PVI. URL: https://www.clinicaltrials.gov; Unique identifier: NCT04056390.

Thrombin receptor PAR4 cross-activates the tyrosine kinase c-met in atrial cardiomyocytes.

Thrombin supports coagulation-independent inflammation via protease-activated receptors (PAR). PAR4 is specifically increased in obese human atria, correlating with NLRP3 inflammasome activation. PAR4-mediated NLRP3 inflammasome activation in atrial cardiomyocytes is not known, nor have signaling partners been identified. Thrombin transactivates the hepatocyte growth factor receptor in some cancer cells, so we examined PAR4/c-met cross-talk in atrial cardiomyocytes and its possible significance in obesity. Cardiomyocytes from right atrial appendages (RAA) of obese patients expressed more PAR1 and PAR4 compared to non-obese. In HL-1 atrial cardiomyocytes, thrombin induced caspase-1 auto-activation and IL-1β maturation; IL-1β secretion was evoked by PAR4-activating peptide (AP), but not PAR1-AP. PAR4-AP additionally increased phosphorylated CaMKII-Thr287, mTOR-Ser2481, and Akt-Ser473 while suppressing AMPK-Thr172 phosphorylation. Total kinase levels were largely unaltered. PAR4AP rapidly increased phosphorylated c-met in HL-1 cells and over time also transcriptionally upregulated c-met. The c-met inhibitor SGX-523 abrogated the effects of PAR4-AP on CaMKII/AKT/mTOR phosphorylation but did not affect PAR4-stimulated IL-1β production. Obese human RAA contained more IL-1β, phospho-c-met, and phospho-mTOR than non-obese RAA; CamKII phosphorylation was not modified. Atria from high-fat diet (HFD) versus chow-fed mice also contained more IL-1β, together with higher myeloperoxidase activity, Acta2 mRNA total and phosphorylated c-met; these increases were blunted in PAR4-/- HFD-fed mice. Thrombin cross-activates c-met via PAR4 in atrial cardiomyocytes. Transactivated c-met contributes partially to PAR4-mediated signaling, but NLRP3 inflammasome activation appears to be largely independent of c-met. Abundance of PAR4 and activated c-met increases with obesity, providing therapeutic targets for management of adiposity-driven AF.

PDE8 Inhibition and Its Impact on ICa,L in Persistent Atrial Fibrillation: Evaluation of PDE8 as a Potential Drug Target.

Atrial fibrillation (AF) poses a significant therapeutic challenge with drug interventions showing only limited success. Phosphodiesterases (PDE) regulate cardiac electrical stability and may represent an interesting target. Recently, PDE8 inhibition was proposed as an antiarrhythmic intervention by increasing L-type Ca2+ current (ICa,L) and action potential duration (APD). However, the effect size of PDE8 inhibition on ICa,L and APD seems discrepant and effects on force are unknown. We investigated the impact of PDE8 inhibition on force using PF-04957325 in right atrial appendages, obtained from patients in sinus rhythm (SR) and with persistent AF (peAF) undergoing cardiac surgery. A computational model was employed to predict the effects of PDE8 inhibition on APD in SR and peAF. Results showed no increase in force after exposure to increasing concentrations of the PDE8 inhibitor PF-04957325 in either SR or peAF tissues. Furthermore, PDE8 inhibition did not affect the potency or efficacy of norepinephrine-induced inotropic effects in either group. Arrhythmic events triggered by norepinephrine were observed in both SR and peAF, but their frequency remained unaffected by PF-04957325 treatment. Computational modeling predicted that the reported increase in ICa,L induced by PDE8 inhibition would lead to substantial APD prolongation at all repolarization states, particularly in peAF. Our findings indicate that PDE8 inhibition does not significantly impact force or arrhythmogenicity in human atrial tissue.

Distinctive Deposition Patterns of Sporadic Transthyretin-Derived Amyloidosis in the Atria: A Forensic Autopsy-Based Study.

Left-to-right differences in the histopathologic patterns of transthyretin-derived amyloid (ATTR) deposition in the atria of older adults have not yet been investigated. Hence, this study evaluated heart specimens from 325 serial autopsy subjects. The amount of ATTR deposits in the seven cardiac regions, including both sides of atria and atrial appendages, was evaluated semiquantitatively. Using digital pathology, we quantitatively evaluated the immunohistochemical deposition burden of ATTR in the myocardium. We identified 20 sporadic ATTR cardiac amyloidosis cases (nine males). All patients had ATTR deposition in the left atrial regions of the myocardium. In the semiquantitative analysis, 14 of the 20 cases showed more severe ATTR deposition on the left atrial regions than on the right side, with statistically significant differences in the pathology grading (p < 0.01 for both the atrium and atrial appendage). Quantitative analysis further supported the difference. Moreover, six had ATTR deposition in the epineurium and/or neural fibers of the atria. Cluster analysis revealed that ATTR deposition in the myocardium was significantly more severe in males than in females. The heterogeneous distribution of amyloid deposits between atria revealed in this study may impair the orderly transmission of the cardiac conduction system and induce arrhythmias, which may be further aggravated by additional neuropathy in the advanced phase. This impairment could be more severe among males. These findings emphasize that atrial evaluation is important for individuals with sporadic ATTR cardiac amyloidosis, particularly for early detection.

Empagliflozin and dapagliflozin decreased atrial monoamine oxidase expression and alleviated oxidative stress in overweight non-diabetic cardiac patients.

The sodium-glucose-cotransporter 2 inhibitors (SGLT2i) are the blockbuster antidiabetic drugs that exert cardiovascular protection via pleiotropic effects. We have previously demonstrated that empagliflozin decreased monoamine oxidase (MAO) expression and oxidative stress in human mammary arteries. The present study performed in overweight, non-diabetic cardiac patients was aimed to assess whether the two widely prescribed SGLT2i decrease atrial MAO expression and alleviate oxidative stress elicited by exposure to angiotensin 2 (ANG2) and high glucose (GLUC). Right atrial appendages isolated during cardiac surgery were incubated ex vivo with either empagliflozin or dapagliflozin (1, 10µm, 12h) in the presence or absence of ANG2 (100nm) and GLUC (400mg/dL) and used for the evaluation of MAO-A and MAO-B expression and ROS production. Stimulation with ANG2 and GLUC increased atrial expression of both MAOs and oxidative stress; the effects were significantly decreased by the SGLT2i. Atrial oxidative stress positively correlated with the echocardiographic size of heart chambers and negatively with the left ventricular ejection fraction. In overweight patients, MAO contributes to cardiac oxidative stress in basal conditions and those that mimicked the renin-angiotensin system activation and hyperglycemia and can be targeted with empagliflozin and dapagliflozin, as novel off-target class effect of the SGLT2i.

Histological evaluation of cardiac remodelling in equine athletes

Approximately 1–2 per 100,000 young athletes die from sudden cardiac death (SCD) and extreme exercise may be associated with myocardial scar and arrhythmias. Racehorses have a high prevalence of atrial fibrillation (AF) and SCD but the presence of myocardial scar and inflammation has not been evaluated. Cardiac tissues from the left (LAA) and right (RAA) atrial appendages, left ventricular anterior (LVAPM) and posterior (LVPPM) papillary muscles, and right side of the interventricular septum (IVS-R) were harvested from racehorses with sudden cardiac death (SCD, n = 16) or other fatal injuries (OFI, n = 17), constituting the athletic group (ATH, n = 33), and compared to sedentary horses (SED, n = 10). Horses in the ATH group had myocyte hypertrophy at all sites; increased fibrosis at all sites other than the LAA; increased fibroblast infiltration but a reduction in the overall extracellular matrix (ECM) volume in the RAA, LVAPM, and IVS-R compared to SED horses. In this horse model, athletic conditioning was associated with myocyte hypertrophy and a reduction in ECM. There was an excess of fibrocyte infiltration and focal fibrosis that was not present in non-athletic horses, raising the possibility of an exercise-induced pro-fibrotic substrate.

Polycystin-1 loss of function increases susceptibility to atrial fibrillation through impaired DNA damage response.

The increasing prevalence of atrial fibrillation (AF) and chronic kidney diseases highlights the need for a deeper comprehension of the molecular mechanisms linking them. Mutations in PKD1, the gene encoding Polycystin-1 (PKD1 or PC1), account for 85% of autosomal dominant polycystic kidney disease (ADPKD) cases. This disease often includes cardiac complications such as AF. In cardiomyocytes, PC1 deletion reduces hypertrophic response to pressure overload but promotes baseline ventricular dysfunction, while deletion in fibroblasts ameliorates post-myocardial infarction fibrosis. Despite its known cardiac impact, the role of PC1 in atrial cardiomyocytes and arrhythmias is less understood. Here, we sought to investigate the role of PC1 in AF. We used intracardiac programmed stimulation and optical mapping to evaluate AF inducibility in two mouse models, Pkd1 R3277C, which recapitulates human ADPKD progression, and cardiomyocyte-specific Pkd1 deletion, and their respective controls. Isolated adult mouse atrial cardiomyocytes, human iPSC-derived atrial cardiomyocytes (hiPSC-aCM), and HL-1 cells served as in vitro cellular models. Molecular mechanisms were evaluated using optical mapping and molecular and biochemical approaches. Loss-of-function PC1 mutations significantly increased AF susceptibility in vivo and facilitated local reentry in ex vivo left atrial appendages. Comprehensive in vitro experiments supported a direct effect of PC1 in atrial cardiomyocytes. PC1-deficient monolayers exhibited increased arrhythmic events, escalating into reentrant spiral waves post-tachypacing. Transcriptomics analysis revealed PC1-dependent regulation of DNA repair, with PC1 deficiency leading to increased DNA damage under stress. PARP1 inhibitors or nicotinamide riboside, which counteract DNA damage-related metabolic consequences, reduced in vitro arrhythmias PC1-deficient monolayers. Overexpression of the C-terminus of PC1 had the opposite effects in DNA repair genes, suggesting its regulatory effects in atrial cardiomyocytes through retinoblastoma/E2F. Analyses of human atrial tissue from non-ADPKD patients showed reduced levels of mature PC1, suggesting a broader relevance of impaired PC1 in AF. Impaired PC1 increases in vivo AF inducibility under programmed electrical stimulation and promotes in vitro arrhythmias in hiPSC-aCM and HL-1 cells. Our findings indicate that PC1 protects against DNA damage to reduce AF susceptibility.

Left atrial reservoir strain is a marker of atrial fibrotic remodeling in patients undergoing cardiovascular surgery: Analysis of gene expression.

Left atrial strain (LAS) measured by two-dimensional speckle tracking echocardiography (2DSTE) is considered to be a marker of LA structural remodeling, but it remains unsettled. We investigated the potential usefulness and clinical relevance of LAS to detect atrial remodeling including fibrosis by analyzing gene expression in cardiovascular surgery patients. Preoperative 2DSTE was performed in 131 patients (92 patients with sinus rhythm [SR] patients including paroxysmal AF [PAF], 39 atrial fibrillation [AF]) undergoing cardiovascular surgery. Atrial samples were obtained from the left atrial appendages, and mRNA expression level was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) in 59 cases (24 PAF, 35 AF). Mean value of left atrial reservoir strain (mLASr) correlated with left atrial volume index (LAVI), and left atrial conduit strain (mLAScd). mLASr also correlated with left atrial contractile strain (mLASct) in SR patients including PAF. mLASr was significantly lower, and LAVI was higher, in the AF group, compared with SR patients including PAF. The expression of COL1A1 mRNA encoding collagen type I α1 significantly increased in AF patients (p = 0.031). mLASr negatively correlated with COL1A1 expression level, and multivariate regression analysis showed that mLASr was an independent predictor of atrial COL1A1 expression level, even after adjusting for age, sex, and BMI. But, neither mLAScd / mLASct nor LAVI (bp) correlated with COL1A1 gene expression. The expression level of COL1A1 mRNA strongly correlated with ECM-related genes (COL3A1, FN1). It also correlated ECM degradation-related genes (MMP2, TIMP1, and TIMP2), pro-fibrogenic cytokines (TGFB1 encoding TGFβ1, END1, PDGFD, CTGF), oxidant stress-related genes (NOX2, NOX4), ACE, inflammation-related genes (NLRP, IL1B, MCP-1), and apoptosis (BAX). Among the fibrosis-related genes examined, univariable regression analysis showed that log (COL1A1) was associated with log (TGFB1) (adjusted R2 = 0.685, p<0.001), log (NOX4) (adjusted R2 = 0.622, p<0.001), log (NOX2) (adjusted R2 = 0.611, p<0.001), suggesting that TGFB1 and NOX4 was the potent independent determinants of COL1A1 expression level. mLASr negatively correlated with the ECM-related genes, and fibrosis-related gene expression level including TGFB1, NOX2, and NLRP3 in PAF patients. PAF patients with low mLASr had higher expression of the fibrosis-related gene expression, compared with those with high mLASr. These results suggest that LASr correlates with atrial COL1A1 gene expression associated with fibrosis-related gene expression. Patients with low LASr exhibit increased atrial fibrosis-related gene expression, even those with PAF, highlighting the utility of LAS as a marker for LA fibrosis in cardiovascular surgery patients.

Multiphase patient-specific simulations to study fibrillation-induced thrombosis in the left atrial appendage

Thrombosis commonly develops in the left atrial appendage of patients with atrial fibrillation. The formation of blood clots in atrial appendage is a complex process, which depends on the shape of the appendage, the velocity of blood flow, the concentrations of red blood cells, platelets, and coagulation, among other factors. In this work, we present a new methodology to identify the key factors contributing to clot formation in the left atrial appendage during atrial fibrillation. The new methodology combines computed tomography imaging, computational fluid dynamics, mesh processing, and multiphase thrombosis modeling. We begin by running simulations to investigate flow patterns inside the left atrial appendages with realistic geometries. Our simulations suggest that at the entrance of the left atrial appendage, the flow forms vortices, which can intrude inside the appendage depending on the phases of the cardiac cycle. Next, we introduce blood coagulation and consider different scenarios corresponding to physiological values of blood flow velocity, geometry of the left atrial appendage, and hematocrit values. Numerical results suggest that the chances of clot formation are higher in the “cactus” geometry than in the “chicken-wing” one, in agreement with the literature. Furthermore, they suggest that slower flow circulation facilitates the development of a clot in the depth of the left atrial appendage. Slower blood movement also favors the procoagulant activity of platelets, while faster flow circulation enhances the procoagulant effect from erythrocytes. Finally, our simulations show that increased hematocrit upregulates the generation of fibrin polymer, regardless of flow velocity.

Identification and Analysis of Atrial-Bronchial-Abdominal Disharmony in Patients with Isomeric Atrial Appendages.

Ideally, the morphology of atrial appendages should solely be used to identify and differentiate patients with isomeric right and left atrial appendages. However, in clinical practice, the segregation is often indirectly based on the arrangement of thoraco-abdominal structures. The correlation between thoraco-abdominal arrangement and atrial appendages, however, is imperfect. In this study, we sought to clarify the cardiovascular malformations in patients with isomeric atrial appendages with an emphasis on atrial-thoracic-abdominal disharmony. A retrospective review of all patients who underwent cardiac CT angiography between January 2014 and June 2023 and identified to have isomeric atrial appendages was performed. Of the 366 cases (median age: 2years [interquartile range: 11months-7years]), 247 (67.5%) patients had isomeric right atrial appendages while 119 (32.5%) patients had isomeric left atrial appendages. In 316 (86.3%) patients, the thoraco-abdominal arrangement was as per atrial appendage morphology while the remaining 50 (13.6%) patients had disharmonious patterns. Compared to isomeric left atrial appendages, the disharmonious pattern was more frequent with isomeric right atrial appendages (5.9% vs. 17.4%; p 0.003). Irrespective of the type of isomerism, disharmony was mostly confined to the level of the abdomen. Not all patients with isomeric atrial appendages have a harmonious thoraco-abdominal arrangement. The atrial-bronchial-abdominal disharmony is more frequent with isomeric right atrial appendages and is mostly present at the level of the abdomen. A detailed sequential segmental analysis with an independent description of each organ system is, therefore, essential for the complete evaluation of patients with isomeric atrial appendages.

Small non-coding RNA signatures in atrial appendages of patients with atrial fibrillation.

The development of high-throughput technologies has enhanced our understanding of small non-coding RNAs (sncRNAs) and their crucial roles in various diseases, including atrial fibrillation (AF). This study aimed to systematically delineate sncRNA profiles in AF patients. PANDORA-sequencing was used to examine the sncRNA profiles of atrial appendage tissues from AF and non-AF patients. Differentially expressed sncRNAs were identified using the R package DEGseq 2 with a fold change >2 and p < 0.05. The target genes of the differentially expressed sncRNAs were predicted using MiRanda and RNAhybrid. Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. In AF patients, the most abundant sncRNAs were ribosomal RNA-derived small RNAs (rsRNAs), followed by transfer RNA-derived small RNAs (tsRNAs), and microRNAs (miRNAs). Compared with non-AF patients, 656 rsRNAs, 45 miRNAs, 191 tsRNAs and 51 small nucleolar RNAs (snoRNAs) were differentially expressed in AF patients, whereas no significantly differentially expressed piwi-interacting RNAs were identified. Two out of three tsRNAs were confirmed to be upregulated in AF patients by quantitative reverse transcriptase polymerase chain reaction, and higher plasma levels of tsRNA 5006c-LysCTT were associated with a 2.55-fold increased risk of all-cause death in AF patients (hazard ratio: 2.55; 95% confidence interval, 1.56-4.17; p < 0.001). Combined with our previous transcriptome sequencing results, 32 miRNA, 31 snoRNA, 110 nucleus-encoded tsRNA, and 33 mitochondria-encoded tsRNA target genes were dysregulated in AF patients. GO and KEGG analyses revealed enrichment of differentially expressed sncRNA target genes in AF-related pathways, including the 'calcium signaling pathway' and 'adrenergic signaling in cardiomyocytes.' The dysregulated sncRNA profiles in AF patients suggest their potential regulatory roles in AF pathogenesis. Further research is needed to investigate the specific mechanisms of sncRNAs in the development of AF and to explore potential biomarkers for AF treatment and prognosis.

Characterization of senescence in human cardiac fibroblasts

Abstract Funding Acknowledgements None. Background Recent studies suggest that senescence of cardiac fibroblasts (cFib) plays a significant role in cardiac aging and disease. However, senescence of cultured cFib in response to classical pro-senescent stimuli has not been characterized yet. Purpose We thoroughly studied oncogene- and stress-induced senescence, as well as replicative senescence, in primary human cFib. Methods Human cFib were isolated from specimens of right atrial appendages, collected during cardiac surgery with extracorporeal circulation. These cells were subjected to oncogene-induced senescence by overexpression of HRasV12, were exposed to ionizing radiation (IR, 5 Gy) to recapitulate stress-induced senescence, and were passaged until replicative senescence was reached (P12-P14). Senescence was assessed by staining for senescence associated (SA) β-galactosidase and EdU and by RT-PCR for CDKN1A, CDKN2A, and LamB1. Results The main results are summarized in the graphical abstract. HRasV12 overexpression, IR, and prolonged culturing consistently caused senescence of cFib: in fact, all senescent models showed a significant increase in SA-β-gal activity and CDKN1A expression and a decrease in EdU uptake and LamB1 expression over control cells. Interestingly, cFib with HRasV12 overexpression exhibited a peculiar morphological phenotype with vacuoles and showed higher levels of the typical markers of senescence compared to control and to IR-treated or extensively passaged cFib. Moreover, only cFib with HRasV12 overexpression showed a strong downregulation of CDKN2A. Conclusions Cultured primary human cFib undergo senescence in response to established inducers of senescence. HRasV12 overexpression uniquely causes vacuolization of senescent cFib, which is currently being further characterized by transmission electron microscopy.

2-Aminoethoxydiphenyl borate prolongs atrial and shortens ventricular action potential duration through modulation of ion channels in the sarcolemma

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): PPP allowance, Health holland ZonmW Background 2-Aminoethoxydiphenyl Borate (2-APB) has been shown to prevent atrial fibrillation in animal models, supposedly through its inhibitory action on the intracellular IP3 receptor and subsequent calcium regulation. However, 2-APB may modulate other ion channels or transporters and the cellular mechanisms associated with the prevention of atrial fibrillation are not well established. We aimed at unravelling the cellular effects of 2-APB on atrial and ventricular action potential and currents. Methods Rabbit (New Zealand White) left atrial and ventricular cardiomyocytes were isolated from Langendorff-perfused hearts by enzymatic dissociation. Adult human atrial cardiomyocytes were obtained by enzymatic dissociation from left atrial appendages (LAA) routinely removed from AF patients undergoing minimally invasive surgical pulmonary vein isolation. Action potentials (APs) and K+ currents were recorded at 36.5 °C with the amphotericin-B-perforated or whole cell patch clamp technique. RNA was isolated from left atrial and ventricular tissue and sequenced on an Illumina HiSeq4000 platform. Results In rabbit atrial cardiomyocytes, extracellular application of 5 uM 2-APB had no effect on action potential duration (APD), but extracellular application of 50 uM 2-APB prolonged the APD by 40% and inhibited an outward K+-current. Inversely, extracellular application of 5 uM 2-APB shortened the APD of rabbit left ventricular cardiomyocytes by 20% while extracellular application 50 uM 2-APB did not prolong the APD. Additionally, 2-APB did not modulate K+ currents in ventricular rabbit cardiomyocytes. Intracellular application (in the patch pipet) of 50 uM 2-APB did not have any effects on atrial APD, suggesting that IP3 receptor inhibition is not involved. However, subsequent application of 50 uM 2-APB in the bath prolonged the APD by 40% indicating that 2-APB prolongs atrial APD by inhibiting one or more K+ channel types in the sarcolemmal membrane from the extracellular site. RNA sequencing analysis revealed potential targets channel including TRP and Kv channels differentially expressed in atria versus ventricle. In human atrial cardiomyocytes from AF patients, application of 50 uM 2-APB prolonged APD by 25%. Conclusions 2-APB prolongs rabbit and human atrial APD and shortens rabbit ventricular APD in a concentration dependent manner, most likely due to modulation of sarcolemmal K+ channels, differentially expressed in the two cell types. Pharmacological studies with 2-APB may reveal novel cellular mechanisms identifying potential new targets for the treatment of atrial fibrillation.

Activated factor X stimulates atrial endothelial cells and tissues to promote remodelling responses through AT1R/NADPH oxidases/SGLT1/2.

Atrial fibrillation (AF), the most common cardiac arrhythmia favouring ischemic stroke and heart failure involves left atrial remodelling, fibrosis and a complex interplay between cardiovascular risk factors. This study examined whether activated factor X (FXa) induces pro-remodelling and pro-fibrotic responses in atrial endothelial cells (AECs) and human atrial tissues and determined the underlying mechanisms. AECs collected from porcine hearts and human right atrial appendages (RAA) from patients undergoing heart surgery. Protein expression levels were assessed by Western blot and immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and NO using fluorescent probes, thrombin and angiotensin II generation by specific assays, fibrosis by Sirius red staining and senescence by senescence-associated beta-galactosidase (SA-β-gal) activity. In AECs, FXa increased ROS formation, senescence (SA-β-gal activity, p53, p21), angiotensin II generation and the expression of pro-inflammatory (VCAM-1, MCP-1), pro-thrombotic (tissue factor), pro-fibrotic (TGF-β and collagen-1/3a) and pro-remodelling (MMP-2/9) markers whereas eNOS levels and NO formation were reduced. These effects were prevented by inhibitors of FXa but not thrombin, protease-activated receptors antagonists (PAR-1/2) and inhibitors of NADPH oxidases, ACE, AT1R, SGLT1/SGLT2. FXa also increased expression levels of ACE1, AT1R, SGLT1/2 proteins which were prevented by SGLT1/2 inhibitors. Human RAA showed tissue factor mRNA levels that correlated with markers of endothelial activation, pro-remodelling and pro-fibrotic responses and SGLT1/2 mRNA levels. They also showed protein expression levels of ACE1, AT1R, p22phox, SGLT1/2, and immunofluorescence signals of nitrotyrosine and SGLT1/2 colocalized with those of CD31. FXa increased oxidative stress levels which were prevented by inhibitors of the AT1R/NADPH oxidases/SGLT1/2 pathway. FXa promotes oxidative stress triggering premature endothelial senescence and dysfunction associated with pro-thrombotic, pro-remodelling and pro-fibrotic responses in AECs and human RAA involving the AT1R/NADPH oxidases/SGLT1/2 pro-oxidant pathway. Targeting this pathway may be of interest to prevent atrial remodelling and the progression of atrial fibrillation substrate.

Atrial Fibrosis and Inflammation in Postoperative Atrial Fibrillation: Comparative Effects of Amiodarone, Colchicine, or Exosomes

BackgroundExtracellular vesicles (EVs) isolated from human heart-derived cells have shown promise in suppressing inflammation and fibroblast proliferation. However, their precise benefits in atrial fibrillation (AF) prevention and the role of their antifibrotic/anti-inflammatory properties remain unclear. ObjectivesThe purpose of this study was to conduct a head-to-head comparison of antiarrhythmic strategies to prevent postoperative AF using a rat model of sterile pericarditis. Specifically, we aimed to assess the efficacy of amiodarone (a classic antiarrhythmic drug), colchicine (an anti-inflammatory agent), and EVs derived from human heart-derived cells, which possess anti-inflammatory and antifibrotic properties, on AF induction, inflammation, and fibrosis progression. MethodsHeart-derived cells were cultured from human atrial appendages under serum-free xenogen-free conditions. Middle-aged Sprague Dawley rats were randomized into different groups, including sham operation, sterile pericarditis with amiodarone treatment, sterile pericarditis with colchicine treatment (2 dose levels), and sterile pericarditis with intra-atrial injection of EVs or vehicle. Invasive electrophysiological testing was performed 3 days after surgery before sacrifice. ResultsSterile pericarditis increased the likelihood of inducing AF. Colchicine and EVs exhibited anti-inflammatory effects, but only EV treatment significantly reduced AF probability, whereas colchicine showed a positive trend without statistical significance. EVs and high-dose colchicine reduced atrial fibrosis by 46% ± 2% and 26% ± 2%, respectively. Amiodarone prevented AF induction but had no effect on inflammation or fibrosis. ConclusionsIn this study, both amiodarone and EVs prevented AF, whereas treatment with colchicine was ineffective. The additional anti-inflammatory and antifibrotic effects of EVs suggest their potential as a comprehensive therapeutic approach for AF prevention, surpassing the effects of amiodarone or colchicine.

Juxtaposed Atrial Appendages: Implications for Cardiac Electronic Device Implants

Juxtaposed Atrial Appendages: Implications for Cardiac Electronic Device Implants