Human Atrial Appendage Research Articles

Secretomes from human epicardial adipocytes modulate atrial lymphatics and atrial fibrosis

Abstract Background Cardiac lymphatics maintains myocardial fluid and immune cell homeostasis. Downregulation of cardiac lymphatics leads to cardiac dysfunction through impairment of inflammation and edema. On the other hand, it is well known that adipose tissue accumulation causes lymphatic dysfunction and impairs lymphedema. Purpose We evaluated the expression of atrial lymphatics in human left atrial appendage (LAA) histologically and biologically. Subsequently, we examined secretomes from epicardial adipose tissue (EAT)-derived adipocytes and validated if they change atrial lymphatics expression using ex vivo Organo-culture system. Methods Human LAA samples were collected from forty-two patients during cardiovascular surgery. They were categorized into the sinus rhythm (SR) group (n=16, 73.1±10.4 years), the paroxysmal AF (PAF) group (n=13, 69.8±12.6 years) and the persistent AF (PeAF) group (n=13, 71.2±6.5 years). Human EAT were also excised from patients with and without AF (SR; n=5, AF; n=11) during cardiovascular surgery. Preadipocytes were extracted from EAT by homogenization and collagenase treatment, and cultured to confluence. After differentiation and maturation to adipocytes, cell culture supernatant (CCS) was collected, and was dropped onto the epicardial side of left atrial tissues excised from 8 weeks old male rats for 7 days using an ex vivo organo-culture system. Results In human LAA samples, mRNA expression of lymphangiogenesis-related genes (LYVE1, PROX1, VEGFC, and VEGFR3) was significantly lower in the PeAF group compared to the SR group (p=0.0370, <0.0001, 0.0157, and 0.0009). The number of LYVE1-positive atrial lymphatic endothelial cells (LECs) was also significantly lower in the PeAF group compared to the SR group and the PAF group (p=0.0186 and 0.038). There was a negative correlation between the number of LYVE1-positive atrial LECs and the area percentage of atrial myocardial fibrosis (r=-0.5350, p<0.001). In the experiment using organo-culture system, lymphangiogenesis-related genes expression (Lyve1, Prox1, Vegfc, and Vegfr3) of rat atria treated with CCS of adipocytes from AF patients was significantly lower than those from SR patients (p=0.0391, 0.0100, 0.0034, and 0.0338). Furthermore, the CCS of adipocytes from AF patients contained higher protein levels of leptin, which is known to suppress lymphangiogenesis (p=0.0357). Leptin treatment (100ng/ml) on rat atria also decreased lymphangiogenesis-related genes expression such as Lyve1, Prox1, Vegfc, and Vegfr3 (p=0.0413, < 0.0001, <0.0001, and 0.0190). Conclusions Our study with human LAA demonstrated that atrial lymphatics interact with atrial myocardial fibrosis/progression of AF. Furthermore, our findings indicate that secretomes from EAT-derived adipocytes regulate atrial lymphatics expression, and leptin plays an important role in modulating atrial lymphangiogenesis.

Accumulated β-catenin is associated with human atrial fibrosis and atrial fibrillation

BackgroundAtrial fibrillation (AF) is associated with increased risk of stroke and mortality. It has been reported that the process of atrial fibrosis was regulated by β-catenin in rats with AF. However, pathophysiological mechanisms of this process in human with AF remain unclear. This study aims to investigate the possible mechanisms of β-catenin in participating in the atrial fibrosis using human right atrial appendage (hRAA) tissues .MethodsWe compared the difference of β-catenin expression in hRAA tissues between the patients with AF and sinus rhythm (SR). The possible function of β-catenin in the development of AF was also explored in mice and primary cells.ResultsFirstly, the space between the membrane of the gap junctions of cardiomyocytes was wider in the AF group. Secondly, the expression of the gap junction function related proteins, Connexin40 and Connexin43, was decreased, while the expression of β-catenin and its binding partner E-cadherin was increased in hRAA and cardiomyocytes of the AF group. Thirdly, β-catenin colocalized with E-cadherin on the plasma membrane of cardiomyocytes in the SR group, while they were dissociated and accumulated intracellularly in the AF group. Furthermore, the expression of glycogen synthase kinase 3β (GSK-3β) and Adenomatous Polyposis Coli (APC), which participated in the degradation of β-catenin, was decreased in hRAA tissues and cardiomyocytes of the AF group. Finally, the development of atrial fibrosis and AF were proved to be prevented after inhibiting β-catenin expression in the AF model mice.ConclusionsBased on human atrial pathological and molecular analyses, our findings provided evidence that β-catenin was associated with atrial fibrosis and AF progression.

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.

Sirt1 Inhibits Atrial Fibrosis by Downregulating the Expression of the Transforming Growth Factor-β1/Smad Pathway.

Atrial fibrosis is an important factor leading to atrial fibrillation, and the transforming growth factor-β1/Smad pathway is a key factor in inducing atrial fibrosis. Sirt1 is a member of the histone deacetylase (sirtuin) family, and recent studies have proven its cardioprotective effects. This study explored the effect of Sirt1 on atrial fibrosis through the transforming growth factor-β1/Smad pathway. We analyzed human right atrial appendage tissues and explored the relationship between Sirt1 and atrial fibrosis at the morphological, functional and molecular levels by Masson trichrome staining, immunofluorescence, real-time quantitative polymerase chain reaction and Western blot analysis. Rat atrial fibroblasts were extracted and treated by the Sirt1 agonist resveratrol, inhibitor sirtinol, and recombinant human transforming growth factor-β1 protein. The expression levels of related proteins were detected by Western blot, and the effect on the migration of atrial fibroblasts was detected by wound healing assay. We found that the expression of Sirt1 was reduced in the right atrial appendage tissues of patients with atrial fibrillation, and the degree of fibrosis was increased. In atrial fibroblasts, the activation of Sirt1 could inhibit the expression of transforming growth factor-β1/Smad and reduce the development of fibrosis, while inhibiting Sirt1 reduced its inhibitory effect on the transforming growth factor-β1/Smad pathway. These findings indicate that Sirt1 inhibits atrial fibrosis by downregulating the expression of the transforming growth factor-β1/Smad pathway, and provide potential targets for the treatment of atrial fibrillation.

C-kitpos cells in the human left atrial appendage

BackgroundSubpopulations of myocardial c-kitpos cells have the ability to stimulate regeneration in ischemic heart disease by paracrine effects. The left atrial appendage (LAA), which is easy accessible during cardiac surgery, may represent a perfect source for c-kitpos cell extraction for autologous cell therapies in the living human. So far, frequency and distribution of c-kitpos cells in LAA are unknown. MethodsLAAs of patients who underwent cardiac surgery due to coronary artery disease (coronary artery bypass graft, CABG), valvular heart disease or both and of two body donors were examined. Tissue was fixed in 4 % paraformaldehyde, embedded in paraffin, dissected in consecutive sections and stained for c-kitpos cells. In parallel, grade of fibrosis, amount of fat per section and cells positive for mast cell tryptase were examined. ResultsWe collected 27 LAAs (37.0 % female, mean left ventricular ejection fraction 50.4 %, 63.0 % persistent atrial fibrillation (AF)). Most of the patients underwent combined CABG and valve surgery (51.9 %). C-kitpos cells were detected in 3 different regions: A) Attached to the epicardial fat layer, B) close to vascular structures and C) between cardiomyocytes. C-kitpos cells ranged from 0.05 c-kitpos cells per mm2 to 67.5 c-kitpos cells per mm2. We found no association between number of c-kitpos cells and type of AF, amount of fibrosis or amount of fat. Up to 72 % of c-kitpos cells also showed a positive staining for mast cell tryptase. ConclusionC-kitpos cells are frequent in LAAs of cardiovascular patients with a rather homogenous distribution throughout the LAA. The LAA can therefore be considered as a source for extraction of a reasonable quantity of autologous cardiac progenitor cells in the living human patient.

Changes of ubiquitylated proteins in atrial fibrillation associated with heart valve disease: proteomics in human left atrial appendage tissue.

Correlations between posttranslational modifications and atrial fibrillation (AF) have been demonstrated in recent studies. However, it is still unclear whether and how ubiquitylated proteins relate to AF in the left atrial appendage of patients with AF and valvular heart disease. Through LC-MS/MS analyses, we performed a study on tissues from eighteen subjects (9 with sinus rhythm and 9 with AF) who underwent cardiac valvular surgery. Specifically, we explored the ubiquitination profiles of left atrial appendage samples. In summary, after the quantification ratios for the upregulated and downregulated ubiquitination cutoff values were set at >1.5 and <1:1.5, respectively, a total of 271 sites in 162 proteins exhibiting upregulated ubiquitination and 467 sites in 156 proteins exhibiting downregulated ubiquitination were identified. The ubiquitylated proteins in the AF samples were enriched in proteins associated with ribosomes, hypertrophic cardiomyopathy (HCM), glycolysis, and endocytosis. Our findings can be used to clarify differences in the ubiquitination levels of ribosome-related and HCM-related proteins, especially titin (TTN) and myosin heavy chain 6 (MYH6), in patients with AF, and therefore, regulating ubiquitination may be a feasible strategy for AF.

Sarcomeric network analysis of ex vivo cultivated human atrial appendage tissue using super-resolution microscopy

Investigating native human cardiac tissue with preserved 3D macro- and microarchitecture is fundamental for clinical and basic research. Unfortunately, the low accessibility of the human myocardium continues to limit scientific progress. To overcome this issue, utilizing atrial appendages of the human heart may become highly beneficial. Atrial appendages are often removed during open-heart surgery and can be preserved ex vivo as living tissue with varying durability depending on the culture method. In this study, we prepared living thin myocardial slices from left atrial appendages that were cultured using an air-liquid interface system for overall 10 days. Metabolic activity of the cultured slices was assessed using a conventional methyl thiazolyl tetrazolium (MTT) assay. To monitor the structural integrity of cardiomyocytes within the tissue, we implemented our recently described super-resolution microscopy approach that allows both qualitative and quantitative in-depth evaluation of sarcomere network based on parameters such as overall sarcomere content, filament size and orientation. Additionally, expression of mRNAs coding for key structural and functional proteins was analyzed by real-time reverse transcription polymerase chain reaction (qRT-PCR). Our findings demonstrate highly significant disassembly of contractile apparatus represented by degradation of alpha -actinin filaments detected after three days in culture, while metabolic activity was constantly rising and remained high for up to seven days. However, gene expression of crucial cardiac markers strongly decreased after the first day in culture indicating an early destructive response to ex vivo conditions. Therefore, we suggest static cultivation of living myocardial slices derived from left atrial appendage and prepared according to our protocol only for short-termed experiments (e.g. medicinal drug testing), while introduction of electro-mechanical stimulation protocols may offer the possibility for long-term integrity of such constructs.

Abstract P3126: Atrial Fibrillation Is Associated With Decreased Claudin-5 In Cardiomyocyte

Background: Although it is critically important to understand the underlying molecular and electrophysiological changes that predispose to the induction and maintenance of atrial fibrillation (Af), the underlying mechanism of Af is still poorly defined. Af is characterized as the electrophysiological and membrane integrity abnormality of the atrial cells, and claudin-5, a tight junction protein, may be involved in the pathophysiology of Af, however, the role of claudin-5 in Af is unknown. Methods and Results: Cardiac tissues from the left atrial appendage were obtained from AF patients with rheumatic valvular heart disease undergoing modified radiofrequency ablation maze procedure with approval from local human research ethic review and normal left atrial appendages were obtained from non-AF donors. Western blot, immunofluorescence and transmission electron microscope showed the expression of claudin-5 significantly decreased in cardiomyocytes from left atrium of AF patients compared to non-AF donors. Then proteomics analysis was performed to screen the specific protein expression and signal pathway changed in AF heart tissue vs. non-AF heart tissue, and showed that 83 proteins were downregulated and 102 proteins were upregulated in the left atrial appendage of AF patients. Among them, DAG, CACNA2D2, MYL2 were downregulated and MCU was upregulated. KEGG pathway analysis showed these changes would lead to hypertrophic and dilated cardiomyopathy. Finally, claudin-5 shRNA AAV was injected in mouse left ventricle to knockdown claudin-5 in cardiomyocytes to observe whether change of claudin-5 affects the protein expression in KEGG pathway analysis. We found claudin-5 deficiency caused severe ventricular dilation, ST elevation and arrythmia, and the protein level of DAG, CACNA2D2 and MYL2 were downregulated and MCU was upregulated. Co-immunoprecipitation also confirmed the specific protein-protein interaction between these targeted proteins and claudin-5. Conclusion: We demonstrated that the decrease of claudin-5 in the left atrium of AF patients with rheumatic valvular heart disease. The mechanism of AF might be associated with claudin-5 downregulation-induced decrease of DAG, CACNA2D2, MYL2 and increase of MCU in cardiomyocytes.

Lipopolysaccharide-induced sepsis impairs M2R-GIRK signaling in the mouse sinoatrial node

Sepsis has emerged as a global health burden associated with multiple organ dysfunction and 20% mortality rate in patients. Numerous clinical studies over the past two decades have correlated the disease severity and mortality in septic patients with impaired heart rate variability (HRV), as a consequence of impaired chronotropic response of sinoatrial node (SAN) pacemaker activity to vagal/parasympathetic stimulation. However, the molecular mechanism(s) downstream to parasympathetic inputs have not been investigated yet in sepsis, particularly in the SAN. Based on electrocardiography, fluorescence Ca2+ imaging, electrophysiology, and protein assays from organ to subcellular level, we report that impaired muscarinic receptor subtype 2-G protein-activated inwardly-rectifying potassium channel (M2R-GIRK) signaling in a lipopolysaccharide-induced proxy septic mouse model plays a critical role in SAN pacemaking and HRV. The parasympathetic responses to a muscarinic agonist, namely IKACh activation in SAN cells, reduction in Ca2+ mobilization of SAN tissues, lowering of heart rate and increase in HRV, were profoundly attenuated upon lipopolysaccharide-induced sepsis. These functional alterations manifested as a direct consequence of reduced expression of key ion-channel components (GIRK1, GIRK4, and M2R) in the mouse SAN tissues and cells, which was further evident in the human right atrial appendages of septic patients and likely not mediated by the common proinflammatory cytokines elevated in sepsis.

Activation of Exchange Proteins directly Activated by cAMP (EPAC) lengthens action potential duration by inhibiting potassium currents in atrial cardiomyocytes

IntroductionAtrial fibrillation (AF) is the most frequent sustained cardiac arrhythmia worldwide. Several mechanisms have been described in AF pathogenesis and sustainability such as dysregulation of calcium cycling and action potential (AP) duration. Effectors involved in these dysregulations are potential relevant therapeutic targets. Among them, the Exchange Proteins directly Activated by cAMP (EPAC) has been shown to increase arrhythmogenic activity in ventricular cardiomyocytes by lengthening of AP duration. However, the electrophysiological impact of EPAC at the atrial level remains to be established. ObjectiveTo evaluate the propensity of EPAC activation to induce atrial arrhythmogenic activity and by which mechanisms. MethodMyocytes from human right atrial appendages and wild type (WT) and EPAC1-/- mice atria were dissociated by enzymatic digestion. AP and potassium currents were recorded using the whole cell configuration of the patch clamp technique. EPAC proteins were pharmacologically activated by acute superfusion of 8-CPTAM (10μM), and the selective role of EPAC1 was evaluated by its selective inhibition by superfusion of AM-001 (20μM). ResultsIn mouse atrial myocytes, activation of EPAC by 8-CPTAM significantly increases AP duration at 90% of repolarization (APD90). Interestingly, this effect is higher in WT myocytes than in EPAC1-/-. Moreover, AM-001, which fails to correct AP lengthening in EPAC1-/- myocytes, reverses partially but significantly the effect of 8-CPTAM in WT myocytes. In human atrial myocytes, 8-CPTAM treatment lengthens APD90 by 32.3%. Additionally, in both WT mouse and human atrial myocytes, the treatment by 8-CPTAM induces an inhibition of both peak and sustained components of the K+ currents. Finally, effect of 8-CPTAM on K+ currents seems to be weaker in EPAC1-/- myocytes than in WT. ConclusionOur results show that EPAC activation lengthens AP duration in mouse and human atrial myocytes. Furthermore, inhibition of K+ currents participates in this AP duration modulation. Thus, these data suggest that EPAC proteins might be involved in atrial arrhythmogenic ectopic activity. Nevertheless, the role of each isoform of EPAC in these modulations and EPAC-induced regulation of other ionic currents remain to be specified.

Integrated analysis of the lncRNA-miRNA-mRNA network based on competing endogenous RNA in atrial fibrillation.

Long non-coding RNAs (lncRNAs) play pivotal roles in the transcriptional regulation of atrial fibrillation (AF) by acting as competing endogenous RNAs (ceRNAs). In the present study, the expression levels of lncRNAs of sinus rhythm (SR) patients and AF patients were investigated with transcriptomics technology, and the lncRNA-miRNA-mRNA network based on the ceRNA theory in AF was elaborated. Left atrial appendage (LAA) tissues were obtained from patients with valvular heart disease during cardiac surgery, and they were divided into SR and AF groups. The expression characterizations of differentially expressed (DE) lncRNAs in the two groups were revealed by high-throughput sequencing methods. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed, and the lncRNA-miRNA-mRNA-mediated ceRNA network was constructed. A total of differentially expressed 82 lncRNAs, 18 miRNAs, and 495 mRNAs in human atrial appendage tissues were targeted. Compared to SR patients, the following changes were found in AF patients: 32 upregulated and 50 downregulated lncRNAs; 7 upregulated and 11 downregulated miRNAs; and 408 upregulated and 87 downregulated mRNAs. A lncRNA-miRNA-mRNA network was constructed, which included 44 lncRNAs, 18 miRNAs, and 347 mRNAs. qRT-PCR was performed to verify these findings. GO and KEGG analyses suggested that inflammatory response, chemokine signaling pathway, and other biological processes play important roles in the pathogenesis of AF. Network analysis based on the ceRNA theory identified that lncRNA XR_001750763.2 and Toll-like receptor 2 (TLR2) compete for binding to miR-302b-3p. In AF patients, lncRNA XR_001750763.2 and TLR2 were upregulated, and miR-302b-3p was downregulated. We identified a lncRNA XR_001750763.2/miR-302b-3p/TLR2 network based on the ceRNA theory in AF. The present study shed light on the physiological functions of lncRNAs and provided information for exploring potential treatments for AF.

Cantharidin increases the force of contraction and protein phosphorylation in isolated human atria.

Cantharidin, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), is known to increase the force of contraction and shorten the time to relaxation in human ventricular preparations. We hypothesized that cantharidin has similar positive inotropic effects in human right atrial appendage (RAA) preparations. RAA were obtained during bypass surgery performed on human patients. These trabeculae were mounted in organ baths and electrically stimulated at 1Hz. For comparison, we studied isolated electrically stimulated left atrial (LA) preparations and isolated spontaneously beating right atrial (RA) preparations from wild-type mice. Cumulatively applied (starting at 10 to 30µM), cantharidin exerted a positive concentration-dependent inotropic effect that plateaued at 300µM in the RAA, LA, and RA preparations. This positive inotropic effect was accompanied by a shortening of the time to relaxation in human atrial preparations (HAPs). Notably, cantharidin did not alter the beating rate in the RA preparations. Furthermore, cantharidin (100µM) increased the phosphorylation state of phospholamban and the inhibitory subunit of troponin I in RAA preparations, which may account for the faster relaxation observed. The generated data indicate that PP1 and/or PP2A play a functional role in human atrial contractility.

Abstract 14554: Novel Upstream Targets for Atrial Fibrillation

Introduction: Atrial fibrillation (AF) represents one of the most common arrhythmias seen clinically and is associated with a significant increase in morbidity and mortality, yet, current treatment paradigms have proven largely inadequate. One of the main contributors to the pathophysiology of AF is inflammation. Hence, reduction of inflammation associated atrial remodeling represents a novel therapeutic strategy for the treatment of AF. One of the most biologically important groups of oxylipins (oxygenated lipids) is the eicosanoids that are potent modulators of immune responses and are derived from arachidonic acid, linoleic acid among others, which are metabolized through three pathways including the cytochrome P450 (CYP) pathway. The CYP products are major anti-inflammatory metabolites with several cardioprotective effects. Hypothesis: We hypothesize that lipid metabolites may represent a new therapeutic target for AF Methods: In the cross-sectional study, human atrial appendage specimens and blood from informed and consented patients were obtained in accordance with the approved UC Davis IRB protocol. Metabolomic profiling for over 100 metabolites was assessed from the plasma of patients using LC-MS/MS. We used Functional Connectome, an extension of the singular value decomposition for matrices, to determine how the metabolites were functionally related to each other. The underlying mechanisms of AF were determined using an integrated approach with molecular biology, flow cytometry, and electrophysiology. Results: Metabolomic profiling shows a significant difference in several metabolites from the arachidonic and linoleic acid pathways in the cohort with AF compared to patients in NSR. There was a significant increase in oxidative stress, endoplasmic reticulum stress, inflammatory cytokines, nuclear translocation of NF-κB, and the activation of MAPK and TGF-b pathways from patients with AF compared to NSR cohort. Conclusions: The study provides important insights into the functional relatedness of the metabolites from the CYP pathway in patients with AF and reveals the prospect of arachidonic and linoleic acid metabolites being novel therapeutic targets in the treatment of AF.

Therapeutic knockdown of miR-320 improves deteriorated cardiac function in a pre-clinical model of non-ischemic diabetic heart disease

Non-ischemic diabetic heart disease (NiDHD) is characterized by diastolic dysfunction and decreased or preserved systolic function, eventually resulting in heart failure. Accelerated apoptotic cell death because of alteration of molecular signaling pathways due to dysregulation in microRNAs (miRNAs) plays a significant role in the development of NiDHD. Here, we aimed to determine the pathological role of cardiomyocyte-enriched pro-apoptotic miR-320 in the development of NiDHD. We identified a marked upregulation of miR-320 that was associated with downregulation of its target protein insulin growth factor-1 (IGF-1) in human right atrial appendage tissue in the late stages of cardiomyopathy in type 2 diabetic db/db mice and high-glucose-cultured human ventricular cardiomyocytes (AC-16 cells). In vitro knockdown of miR-320 in high-glucose-exposed AC-16 cells using locked nucleic acid (LNA) anti-miR-320 markedly reduced high-glucose-induced apoptosis by restoring IGF-1 and Bcl-2. Finally, in vivo knockdown of miR-320 in 24-week-old type 2 diabetic db/db mice reduced cardiomyocyte apoptosis and interstitial fibrosis while restoring vascular density. This resulted in partial recovery of the impaired diastolic and systolic function. Our study provides evidence that miR-320 is a late-responding miRNA that aggravates apoptosis and cardiac dysfunction in the diabetic heart, and that therapeutic knockdown of miR-320 is beneficial in partially restoring the deteriorated cardiac function.

Cellular remodeling of human right atrial appendage during aging and myocardial infarction

Cardiac aging is characterized by cardiomyocyte loss, hypertrophy of the remaining cardiomyocytes and replacement fibrosis, leading to diastolic dysfunction. However, mechanisms involved in age-associated cellular loss such as apoptosis, necroptosis, and/or autophagy, remain controversial but are crucial in age-associated pathologies such as non-insulin dependent diabetes mellitus (NIDD) and atrial fibrillation (AF). The objectives of the present study are to identify the mechanisms involved in cardiomyocytes loss and atrial remodeling during aging. Atrial samples (residues of transection of the right atrial appendage for extracorporeal circulation placement) were collected from patients undergoing coronary artery bypass graft from 36 to 83 years old (yo), with or without history of myocardial infarction (MI), (INSERM IRB authorization n C-19-71). Only patients without NIDD and/or AF were studied ( n = 41). After trabeculae dissection and protein extraction, Beclin-1, RIPK3, pro- and cleaved caspase 3 as well as JNK, ERK, AMPK, P38 MAPK and STAT3 pathways were quantified by western blotting in cytosolic fractions and expressed as normalized densitometric score. Phospho-ERK/ERK and phospho-p38 MAPK levels increased with age (factorial ANOVA; respectively P < 0.001 and P < 0.05) but not with MI. In addition, RIPK3 content increased in samples from patients without MI over 70 yo as compared to patients under 55 yo (1.88 ± 0.23 vs. 0.69 ± 0.27; P < 0.05) and was associated with an activation of JNK pathway (phospho-JNK/JNK level: 2.28 ± 0.98 vs. 0.37 ± 0.09; P < 0.05). In contrast, no significant change was observed for Beclin-1 or caspase 3 levels. In patients with MI over 70 yo, as compared to patients of the same age without MI, Beclin-1 (1.26 ± 0.17 vs. 0.83 ± 0.14, P < 0.05) and phospho-STAT3/STAT3 levels (3.09 ± 0.98 vs. 1.18 ± 0.75; P < 0.05) were increased but surprisingly RIPK3 levels were reduced (1.18 ± 0.19 vs. 1.88 ± 0.23; P < 0.05). The age-associated activation of JNK in atria may lead to the loss of Cx43 protein resulting in impaired conduction and contributing to the increasing risk of AF. In addition, the ERK activation, promoting cell survival, is counterbalanced by activation of p38 MAPK, mediating pro-inflammatory cytokine production, and increased RIPK3 suggesting increased necroptosis, without apoptosis. In patients with MI, the high level of Beclin-1 suggests an upregulation of autophagy.

Atrial endomysial fibrosis is associated with sex, atrial fibrillation, heart failure and age in cardiac surgery patients: results from the Catch-Me consortium

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Horizon 2020 Background Risk factors for atrial fibrillation (AF), such as ageing, heart failure and AF itself, enhance AF propensity partly by inducing atrial fibrosis. Atrial endomysial fibrosis, a type of reactive fibrosis occurring between cardiomyocytes, impairs transverse conduction in rapid atrial pacing animal models. The factors underlying transcriptional regulation of endomysial fibrosis are largely unknown. Objective To examine the contributions of age, sex, AF and heart failure to the development of endomysial fibrosis in the context of concurrent pathologies. To study genome-wide transcriptional changes associated with endomysial fibrosis in human left and right atrial appendage biopsies (LAA, n=95; RAA, n=76). Methods An algorithm for automated quantification of endomysial fibrosis following staining with wheat germ agglutinin (WGA) was employed. Linear mixed models were constructed to determine endomysial fibrosis quantity as a function of AF, heart failure, sex, age and four principal components that accounted for potential confounding effects of other clinical characteristics. RNA sequencing was used to study expression changes in the atrial transcriptome associated with endomysial fibrosis. Results Sex, persistent AF, heart failure and age were independently associated with endomysial fibrosis. We identified hundreds (LAA: 386, RAA: 311) of RNA transcripts associated with endomysial fibrosis. None of these associations were independent from the clinical phenotypes. However, explorative gene set enrichment analysis identified association of endomysial fibrosis with gene sets involved in extracellular matrix organization, immune response, cell motility, developmental processes, cardiac muscle contraction and proteostasis in LAA while in RAA only gene sets regulating contractile function were enriched. Conclusion Besides AF, female sex, age and heart failure are associated with endomysial fibrosis in the atria. While abundance of none of the differential genes were independently associated with endomysial fibrosis, gene set enrichment analysis suggests an involvement of extracellular matrix organization, immune response, cell motility, developmental processes and cardiac muscle contraction in endomysial fibrosis.

Differential sodium current remodelling in human left atrial appendage cardiomyocytes in paroxysmal and persistent atrial fibrillation

Abstract Funding Acknowledgements Type of funding sources: None. Background Atrial fibrillation (AF) is characterized by complex electrical, structural and metabolic remodelling. The mechanisms underlying AF progression from paroxysmal to persistent AF are not fully understood, and studies in cardiomyocytes (CMs) at the paroxysmal stage of AF are lacking. Moreover, most studies have so far investigated right atrial appendage CMs, while left atrial appendage (LAA) CMs may be more informative, as AF is predominantly a left atrial disease. Whether and to what extent electrical remodelling during various AF stages also includes alterations of the (late) sodium current (INa), remains unclear. Moreover, the functional relevance of sodium channel isoforms other than the cardiac Nav1.5, such as the "neuronal" isoform SCN10A/NaV1.8, during the various stages of AF is as yet not fully elucidated. Purpose To investigate peak and late INa remodelling in LAA CMs from patients with paroxysmal and persistent AF and patients in sinus rhythm (SR), as well as the potential contribution of NaV1.8-based current. Methods LAA were obtained from patients in SR (N=18) without a history of AF undergoing cardiac surgery, as well as from patients with paroxysmal (N=12) and persistent AF (N=30). Paroxysmal AF was defined as AF episodes terminating spontaneously within seven days and persistent as AF continuing for more than 7 days but less than 1 year. Peak INa, late INa and action potential (AP) properties were investigated through patch-clamp analysis on single LAA CMs, while qPCR was used to assess SCN5A and SCN10A expression levels in LAA tissue. Results In paroxysmal and persistent AF CMs, AP duration was shorter than in SR CMs. Compared to SR, peak INa (Figure 1A) and SCN5A expression (Figure 1B) were significantly decreased in paroxysmal AF, while they were restored to SR levels in persistent AF. Conversely, while late INa was undetectable in SR and paroxysmal AF, it was significantly increased in persistent AF (Figure 2). Peak and late INa Nav1.8-based current was not detected in persistent AF CMs. Similarly, expression of SCN10A was not observed in LAAs at any stage. Conclusions Our study is the first to show that AP shortening already occurs in LAA CMs from paroxysmal AF, potentially contributing to pro-arrhythmia in this early stage of the disease. Moreover, our findings demonstrate that INa is differentially remodelled during various stages of AF, with peak INa reduction occurring during paroxysmal AF, while late INa is increased in persistent AF only. Finally, we have shown that Nav1.8 current does not contribute to the AF-related alterations in INa. These observations are of particular relevance when considering potential pharmacological approaches targeting (late) INa in patients with distinct forms of AF.

BS-515-03 ATRIAL FIBRILLATION ASSOCIATED COMMON INTRONIC RISK VARIANTS IN SYNE2 LEAD TO LOWER EXPRESSION OF NESPRIN-2A1, INCREASED NUCLEAR STIFFNESS AND EARLY AFTER DEPOLARIZATIONS IN CARDIOMYOCYTES

Atrial fibrillation (AF) genome-wide association studies (GWAS) identified significant associations with rs1152591 and linked variant rs1152595 in the SYNE2 gene, encoding the nesprin-2 protein that connects the nuclear membrane with the cytoskeleton. To determine the effects of the AF-associated SNPs on SYNE2 expression and investigate the mechanisms for their association with AF. RNA sequencing was performed on 234 AF human left atrial appendage (LAA) tissues. Human induced pluripotent stem cell-derived cardiomyocytes (iCMs) were used to study the cellular effects of SYNE2 knockdown (KD) or GFP-SYNE2α1 overexpression. Reporter gene vectors was used to study the regulatory roles of the SNPs. Nuclear size and stiffness were measured by immunofluorescent microscopy and atomic force microscopy. Calcium transient assay was performed using epifluorescence microscopy and Fura-2 AM. RNA sequencing of LAA tissues indicated that rs1152591 and rs1152595 were significantly associated with the expression of short SYNE2α1 isoform, without affecting the expression of the full-length SYNE2 mRNA. Risk vs. reference alleles of rs1152591 and rs1152595 had decreased promoter or enhancer activity. SYNE2 siRNA KD or nesprin-2α1 overexpression in human iCMs resulted in ∼12.5% larger nuclear area compared to controls (p<0.001). SYNE2 KD or nesprin-2α1 overexpression led to 57.5% or 33.2% decreases, respectively, in nuclear stiffness compared to controls (p< 0.0001). Nesprin-2α1 overexpression rescued the effects of SYNE2 siRNA KD on calcium transient, significantly decreasing the intracellular calcium concentration (p<0.001) and the incidence of early afterdepolarizations (EADs) (p<0.001). AF-associated SNPs rs1152591 and rs1152595 downregulate the expression of SYNE2α1 in the human LAA. SYNE2α1 has a dominant-negative effect on the nucleus, decreasing nuclear-cytoskeletal connectivity and nuclear stiffness that may protect iCMs from repetitive motion stress, and also has a gain of function activity on the calcium cycling, decreasing EADs that may protect against atrial arrhythmia.

PO-630-01 FAM13B AND RS1717131 ARE THE LIKELY GENE AND SNP RESPONSIBLE FOR THE 5Q31 ATRIAL FIBRILLATION SUSCEPTIBILITY LOCUS

Atrial fibrillation (AF) genome wide association studies (GWAS) have identified an AF susceptibility locus on chr 5q31, between the WNT8A and FAM13B genes. Our prior human left atrial appendage RNA sequencing study found that expression of FAM13B is strongly associated with the AF GWAS lead variant in this locus. However, the regulatory variant controlling FAM13B expression and the mechanism by which FAM13B impacts AF incidence are not well understood.