Background: Atrial adverse remodeling drives the maintenance and progression of atrial fibrillation (AF) through electrical and structural myocardial changes, often accompanied by inflammation. Circulating N-glycans are emerging as biomarkers in inflammatory diseases, yet their role in AF remains undefined. Methods: We profiled the serum N-glycome of 138 patients with AF, non-AF arrhythmias, or sinus rhythm (SR) controls from peripheral venous (PV) and coronary sinus (CS) samples using hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry. Glycan traits associated with AF were identified via logistic regression adjusted for clinical risk factors. Multivariate glycan scores were derived from PV and CS datasets using LASSO regression. In a subset (N=37), plasma proteome profiling was performed with the Olink Reveal panel. Results: Sixty-two glycan peaks were detected; 27 in PV and 8 in CS serum differed significantly between AF and controls. PV and CS glycan scores accurately classified AF, with the PV score correlating with 11 plasma proteins linked to structural remodeling and thrombo-inflammatory processes. The most abundant glycan, A2G2S2 (peak 30), was associated with higher odds of AF after adjusting for confounders (OR 2.22 [95% CI: 1.40?3.75], P = 0.001). CS A2G2S2 correlated with C-reactive protein (R = 0.432, P = 0.0275) and was elevated in patients with left atrial enlargement (P = 0.0354), but unchanged in those with impaired left ventricular ejection fraction or hypertrophy. Conclusion: Integrated profiling of peripheral and cardiac serum identifies novel N-glycosylation signatures in AF. Specific cardiac and circulating N-glycan signatures, including A2G2S2, are associated with AF and reflect inflammation-driven atrial remodeling, highlighting potential mechanistic pathways and biomarker applications. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was funded by the British Heart Foundation (BHF) Intermediate (FS/SBSRF/22/31026) and Senior (FS/SBSRF/22/31033) Fellowships (to S.R.), Oxford BHF Centre of Research Excellence (CRE) grants (to S.R.), the British Research Council (BRC4) NIHR Oxford Biomedical Research Centre grant (to S.R. and C.H.K.Y.), the BHF Clinical Research Training Fellowship (FS/CRTF/25/24786; to C.S.M.), the BHF PhD Studentship (FS/20/7/34992; to L.M.M.), the BHF Studentship grant (FS/4yPhD/F/22/34177; to A.M.J.), and The Royal Society University Research Fellowship (URF\R1\221314; to A.B.). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the South Central-Berkshire B Research Ethics Committee (UK, 18/SC/0404 and 18/SC/0304) at John Radcliffe Hospital, Oxford. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data related to the manuscript is included in the manuscript. The metadata of the patient cohorts that support the findings are available from the first author (C.H.K.Y.) and corresponding author (S.R.) upon request.

Background: Proteins in human plasma serve as critical markers for predicting disease risk and guiding therapeutic development. Current prediction models for atrial fibrillation (AF) largely rely on electronic health records; however, the plasma proteome of patients with AF reflects key biological processes, including inflammation, that are not captured by clinical variables alone. Circulating inflammatory mediators contribute to electrical and structural remodelling in the atria, thereby sustaining the AF phenotype. Identification of plasma proteins associated with AF may therefore improve understanding of the inflammatory and other biological processes underlying AF pathophysiology. Objective: In this study, we profiled the plasma proteome of patients with paroxysmal AF (pxAF), persistent AF (persAF), and non-AF controls using Olink assay technology. Methods: Plasma samples from 30 individuals were analysed with the Olink Reveal panel. Differential expression analysis of normalised protein expression (NPX) values was performed between groups, with differentially expressed proteins (DEPs) defined by P < 0.05. Results: We identified 87 DEPs in pxAF and 107 DEPs in persAF compared with controls. From these, we shortlisted 11 candidate proteins that were upregulated in persAF at baseline and showed reduced expression 12 months after catheter ablation. This subset of proteins is implicated in the regulation of inflammation (CCL23, CXCL10, IL33), metabolism (ALDH3A1, NDUFS6), cell-matrix adhesion (AFAP1L1, LGALS7, SPOCK1), and physiological signalling (NOS1, PROK1, PTH). Conclusion: Collectively, these plasma proteins highlight systemic molecular mechanisms contributing to AF pathogenesis and represent potential AF-specific biomarkers warranting further investigation in larger clinical cohorts and mechanistic studies. ### Competing Interest Statement The authors have declared no competing interest. British Heart Foundation, FS/SBSRF/22/31026, FS/SBSRF/22/31033, FS/CRTF/25/24786 British Heart Foundation Centre of Research Excellence, Oxford, GB NIHR Oxford Biomedical Research Centre

Abstract Background Postoperative atrial fibrillation (POAF) affects up to 50% of cardiac surgery patients and is linked to higher morbidity, longer hospital stays and increased thromboembolic risk. Early identification of at-risk patients remains challenging. Calcitonin (CT), a hormone with anti-fibrotic effects, may serve as a novel biomarker. Methods In 491 patients undergoing elective cardiac surgery, baseline serum CT was measured preoperatively using CT-specific enzyme-linked immunosorbent assay (ELISA). Patients with pre-existing AF were excluded. Associations between CT levels and POAF incidence and onset were evaluated using logistic regression, Cox proportional hazards models, and Kaplan–Meier analysis. Results Among 248 patients with detectable CT levels, 88 patients developed POAF. Higher baseline CT was independently associated with lower risk of POAF (OR 0.68 per 5 pg/ml increase; 95% CI 0.51-0.89; P = 0.009) and delayed arrhythmia onset (adjusted HR 0.941; 95% CI 0.898-0.980, P = 0.0026) after adjusting for covariates. Kaplan–Meier analysis demonstrated a graded relationship between increasing CT levels and reduced cumulative incidence of POAF. In this cohort, baseline CT showed greater discriminative ability than CRP and BNP, although overall model performance remained moderate. Conclusion Higher preoperative circulating CT levels are associated with reduced risk and delayed onset of POAF following cardiac surgery. These findings suggest that calcitonin may have the potential as a biomarker for perioperative risk stratification in POAF. Given the observational design and single-centre setting, further validation in independent cohorts and studies integrating mechanistic insights are warranted.

Atrial fibrillation (AF), the commonest cardiac arrhythmia, is a major contributor to mortality and morbidity. Atrial tissue fibrosis, a hallmark of structural remodelling in AF, is currently incurable and significantly hinders AF-treatment. MicroRNA(miR)-31 is linked to ageing (a key risk factor for AF). Here, we show that AF-patients are characterised by upregulation of miR-31-5p in atrial cardiofibroblasts that negatively regulates the calcitonin receptor (CTR), thereby promoting atrial fibrogenesis and arrhythmia. Specific blockade of miR-31-5p/CTR-mRNA binding with LNA-miRNA-Target-Site-Blocker selectively increases atrial CTR expression and reverses advanced atrial fibrosis and arrhythmogenesis in vivo. These findings suggest a key role for miR-31-5p/CTR binding in promoting atrial fibrosis and arrhythmogenesis, and represents a first example of an RNA-based therapeutic capable of reversing established fibrosis that forms an AF substrate.

BACKGROUND:Peritoneal dialysis (PD) is a life-sustaining therapy for kidney failure, yet its long-term viability is compromised by progressive peritoneal fibrosis in some patients. Currently, reliable treatment options are lacking as the pathogenesis remains poorly understood, with the metabolic underpinnings of fibrotic progression remaining particularly elusive. METHODS:Using a physiologically relevant framework comprising human primary mesothelial cells and a PD fluid-exposed mouse model, we performed bidirectional genetic and pharmacological modulation of branched-chain amino acid catabolism. Integrated proteomic and metabolomic analyses were conducted to investigate downstream metabolic consequences. RESULTS:We identified profound impairment in branched-chain amino acid (BCAA) catabolism, centered on a functional bottleneck at the rate-limiting branched-chain α-ketoacid dehydrogenase complex, as a metabolic hallmark of peritoneal fibrosis. Functional uncoupling of the amino acids from their ketoacid derivatives indicated that branched-chain α-ketoacid (BCKA) burden was more closely linked to the fibrotic phenotype than BCAA abundance alone. Mechanistically, BCKA burden was associated with reduced glucose-6-phosphate dehydrogenase expression and activity, lower NADPH-generating capacity, and increased intracellular oxidant burden. CONCLUSIONS:Together, these findings showed that BCKA burden, rather than BCAA abundance alone, more closely tracked fibrotic responses and was linked to reduced pentose phosphate pathway-associated redox capacity in peritoneal fibrosis.

Gla proteins are a vitamin K-dependent family long studied for their roles in blood clotting and vascular calcification. This review examines their broader involvement in cardiovascular disease, particularly cardiac fibrosis. We highlight that their functions are context-dependent, varying with disease stage and cellular environment, and that several occur independently of γ-carboxylation, challenging the view that this modification is universally required for their function. By comparing fibrosis across the heart, lung, liver, and kidney, we distinguish shared from organ-specific mechanisms. Finally, we discuss prospective therapies, from vitamin K supplementation to receptor-targeted approaches, and the questions that remain before clinical translation.

Background Most mutations causing hypertrophic cardiomyopathy (HCM) affect sarcomeric proteins. Mutations in junctophilin-2 (JPH2) are also implicated, but the underlying mechanisms remain unclear. An A405S variant in JPH2 was identified in a male adolescent patient with interventricular septal (IVS) hypertrophy. The corresponding mouse variant (A399S) produces comparable IVS hypertrophy, establishing causality. Prior data indicated that altered intracellular Ca2+ handling is unlikely to be the primary driver. Methods We generated a CRISPR knock-in mouse model carrying the JPH2-A399S variant. Co-immunoprecipitation mass spectrometry and STED nanoscopy were used to identify JPH2 binding partners. Reactive oxygen species (ROS) were assessed with dihydroethidium in isolated myocytes. Adeno-associated virus serotype 9 (AAV9) was employed to overexpress peroxiredoxin 6 (PRDX6) in mutant hearts. Results PRDX6 was identified as a novel and abundant JPH2-interacting protein. PRDX6 expression was selectively downregulated in the IVS of JPH2-A399S mice and was also reduced in human failing hearts. JPH2-A399S mice exhibited increased ROS levels specifically in IVS myocytes. AAV9-mediated PRDX6 overexpression reversed the IVS hypertrophy phenotype. Conclusions These findings identify PRDX6 downregulation and consequent oxidative stress as a key mechanism driving JPH2-A399S-associated HCM. The results reveal a previously unrecognized role for JPH2 in cardiometabolic regulation and suggest that restoring PRDX6 levels may represent a therapeutic strategy for this form of HCM.

Renal fibrosis is the common outcome in all progressive forms of chronic kidney disease. Unfortunately, the pathogenesis of renal fibrosis remains largely unexplored, among which metabolic reprogramming plays an extremely crucial role in the evolution of renal fibrosis. Ceria nanoparticles (CeNP-PEG) with strong ROS scavenging and anti-inflammatory activities have been applied for mitochondrial oxidative stress and inflammatory diseases. The present study aims to determine whether CeNP-PEG has therapeutic value for renal fibrosis. The unilateral ureteral obstructive fibrosis model was used to assess the therapeutic effects in vivo. Transforming growth factor beta1-induced epithelial-to-mesenchymal transition in HK-2 cells was used as the in vitro cell model. The seahorse bioscience X96 extracellular flux analyzer was used to measure the oxygen consumption rate and extracellular acidification rate. In the present study, CeNP-PEG treatment significantly ameliorated renal fibrosis by increased E-cadherin protein expression, and decreased α-SMA, Vimentin and Fibronectin expression both in vitro and in vivo. Additionally, CeNP-PEG significantly reduced the ROS formation and improved the levels of mitochondrial ATP. The seahorse analyzer assay demonstrated that the extracellular acidification rate markedly decreased, whereas the oxygen consumption rate markedly increased, in the presence of CeNP-PEG. Furthermore, the mitochondrial membrane potential markedly enhanced, hexokinase 1 and hexokinase 2 expression significantly decreased after treatment with CeNP-PEG. CeNP-PEG can block the dysregulated metabolic status and exert protective function on renal fibrosis. This may provide another therapeutic option for renal fibrosis.

The dysregulation of gene expression programs in the human atria during persistent atrial fibrillation (AF) is not completely understood. Here, we reanalyze bulk RNA-sequencing datasets from two studies ( N = 242) and identified 755 differentially expressed genes in left atrial appendages of individuals with persistent AF and non-AF controls. We combined the bulk RNA-sequencing differentially expressed genes with a left atrial appendage single-nucleus multi-omics dataset to assign genes to specific atrial cell types. We found noncoding genes at the IFNG locus ( LINC01479 , IFNG-AS1 ) strongly dysregulated in cardiomyocytes. We defined a gene expression signature potentially driven by androgen receptor signaling in cardiomyocytes from individuals with AF. Cell-type-specific gene expression modules suggested an increase in T cell and a decrease in adipocyte and neuronal cell gene expression in AF. Lastly, we showed that reducing NR4A1 expression, a marker of a poorly characterized human atrial fibroblast subtype, fibroblast activation markers, extracellular matrix remodeling and cell proliferation decreased.

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

Abstract Background Atrial fibrillation (AF) poses a significant therapeutic challenge due to myocardial remodelling, involving both structural and electrical alterations. Recent investigations have shed light on the role of atrial cardiomyocytes (CMs) in secreting Calcitonin (CT), a factor crucial for maintaining atrial tissue integrity. Dysregulated CT secretion in AF showed to contribute to fibrotic tissue production by atrial fibroblasts, exacerbating arrhythmogenesis [1]. However, the direct impact of CT on CM function and arrhythmogenicity remains unclear, driving the objectives of our study. Methods/Results Serum samples from 20 cardiac surgery patients revealed a noteworthy association between higher pre-operative CT levels and a significant ~2.8-fold reduction in the incidence of post-operative AF (poAF). Using freshly isolated atrial guinea pig CMs, confirmed (by qPCR and immunofluorescence) that CMs express CT-receptor (CTR) to enable CT actions in the cell. Functional studies found that CT administration inhibits spontaneous calcium (Ca2+)-release events induced by pacing and decreases the Ca2+ transients amplitude (at 2 Hz; IonOptix μstep system) in fura2-loaded CMs (n=22 cells) in a concentration-dependent manner. Atrial iPSC-CMs transduced with global RGECO sensor (Genetically encoded Ca2+ indicator) and treated with 15 pM CT showed a reduction in the beat rate when paced at 2Hz, and a significantly increased the time to 50% baseline. Evaluation of the expression and phosphorylation of selected Ca2+ handling proteins, which may potentially account for the observed changes in Ca2+ transients, showed no differences in total or phospho-(ser2808) ryanodine receptor, and SERCA2α in response to CT but an increase in phospho-(Ser16) Phospholamban. Conclusion Our findings highlight the effects of CT on atrial CM function. Maintaining physiological CT levels offer a promising approach for managing AF clinically, potentially through the use of already in clinical use CT-analogues.

Atrial fibrillation (AF) is the most common arrhythmia in the world. Human genetics can provide strong AF therapeutic candidates, but the identification of the causal genes and their functions remains challenging. Here, we applied an AF fine-mapping strategy that leverages results from a previously published cross- ancestry genome-wide association study (GWAS), expression quantitative trait loci (eQTLs) from left atrial appendages (LAAs) obtained from two cohorts with distinct ancestry, and a paired RNA sequencing (RNAseq) and ATAC sequencing (ATAC-seq) LAA single-nucleus assay (sn-multiome). At nine AF loci, our co- localization and fine-mapping analyses implicated 14 genes. Data integration identified several candidate causal AF variants, including rs7612445 at GNB4 and rs242557 at MAPT. . Finally, we showed that the repression of the strongest AF-associated eQTL gene, LINC01629, , in human embryonic stem cell-derived cardiomyocytes using CRISPR inhibition results in the dysregulation of pathways linked to genes involved in the development of atrial tissue and the cardiac conduction system.

Osteopenia and osteoporosis are among the most common metabolic bone diseases and represent major public health problems, with sufferers having an increased fracture risk. Diabetes is one of the most common diseases contributing to osteopenia and osteoporosis. However, the mechanisms underlying diabetes-induced osteopenia and osteoporosis remain unclear. Bone reconstruction, including bone formation and absorption, is a dynamic process. Large-conductance Ca2+-activated K+ channels (BK channels) regulate the function of bone marrow-derived mesenchymal stem cells, osteoblasts, and osteoclasts. Our previous studies revealed the relationship between BK channels and the function of osteoblasts via various pathways under physiological conditions. In this study, we reported a decrease in the expression of BK channels in mice with diabetes-induced osteopenia. BK deficiency enhanced mitochondrial Ca2+ and activated classical PINK1 (PTEN induced putative kinase 1)-PRKN/Parkin (parkin RBR E3 ubiquitin protein ligase)-dependent mitophagy, whereas the upregulation of BK channels inhibited mitophagy in osteoblasts. Moreover, SLC25A5/ANT2 (solute carrier family 25 (mitochondrial carrier, adenine nucleotide translocator), member 5), a critical inner mitochondrial membrane protein participating in PINK1-PRKN-dependent mitophagy, was also regulated by BK channels. Overall, these data identified a novel role of BK channels in regulating mitophagy in osteoblasts, which might be a potential target for diabetes-induced bone diseases.

BACKGROUND: Accessory pathways are a common cause of supraventricular tachycardia (SVT) and can lead to sudden cardiac death in otherwise healthy children and adults when associated with Wolff-Parkinson-White syndrome. The goal of this study was to identify genetic variants within a large family with structurally normal hearts affected by SVT and Wolff-Parkinson-White syndrome and determine causality of the gene deficit in a corresponding mouse model. METHODS: Whole exome sequencing performed on 2 distant members of a 3-generation family in which multiple members were affected by SVT or Wolff-Parkinson-White pattern (preexcitation) on ECG identified MRC2 as a candidate gene. Serial electrocardiograms, intracardiac electrophysiology studies, echocardiography, optical mapping studies, and histology were performed on both Mrc2 mutant and WT (wild-type) mice. RESULTS: A rare HET (heterozygous) missense variant c.2969A>G;p.Glu990Gly (E990G) in MRC2 was identified as the leading candidate gene variant segregating with the cardiac phenotype following an autosomal-dominant Mendelian trait segregation pattern with variable expressivity. In vivo electrophysiology studies revealed reentrant SVT in E990G mice. Optical mapping studies in E990G mice demonstrated abnormal retrograde conduction, suggesting the presence of an accessory pathway. Histological analysis of E990G mouse hearts showed a disordered ECM (extracellular matrix) in the annulus fibrosus. Finally, Mrc2 knockdown in human cardiac fibroblasts enhanced accelerated cell migration. CONCLUSIONS: This study identified a rare nonsynonymous variant in the MRC2 gene in individuals with familial reentrant SVT, Wolff-Parkinson-White ECG pattern, and structurally normal hearts. Furthermore, Mrc2 knock-in mice revealed an increased incidence of reentrant SVT and bypass tract formation in the setting of preserved cardiac structure and function.