Background and Aims Aging significantly increases the risk of cardiovascular disease, characterized by progressive cardiac dysfunction. The vascular niche is crucial for maintaining cardiac homeostasis, yet endothelial cell (EC) impairment during aging remains poorly understood. This study investigates epigenetically regulated mechanisms underlying EC-dependent cardiac aging and identifies a critical role for zinc finger and BTB domain-containing protein 16 (ZBTB16).Methods Chromatin accessibility (snATAC-seq) and transcriptomic (snRNA-seq) analyses of aged hearts were performed to define age-related regulatory changes. Functional studies using genetic models were performed to assess cardiac aging phenotypes. In vitro assays examined EC senescence, secretory profiles, and effects of ZBTB16-deficient EC supernatants on fibroblasts, cardiomyocytes, and neurons. Overexpression experiments in vitro and in vivo tested whether ZBTB16 mitigates aging-associated dysfunction.Results Aged hearts exhibited decreased chromatin accessibility and reduced ZBTB16 expression in both humans and mice. Zbtb16 deletion in young mice, including Zbtb16-haploinsufficient and endothelial-specific knockout mice, led to premature aging, diastolic dysfunction, and increased secretion of pro-fibrotic and inflammatory factors. ZBTB16-deficient EC supernatants activated fibroblasts, induced cardiomyocyte hypertrophy, and impaired neuronal sprouting. Overexpression of ZBTB16 reversed these effects in senescent ECs and aged mice and reduced diastolic dysfunction. Mechanistic studies identified nuclear receptor-interacting protein 1 as a downstream target suppressed by ZBTB16, thereby limiting fibroblast activation and pro-fibrotic signalling.Conclusions ZBTB16 preserves endothelial integrity and vascular niche homeostasis, protecting against aging-associated cardiac dysfunction. Its loss promotes EC senescence and fibrosis, whereas restoring its expression may represent a therapeutic strategy to improve cardiac function and reduce cardiovascular disease risk during aging.

TET2-driven clonal hematopoiesis has been associated with reduced Alzheimer's disease risk, but mechanisms in humans remain unclear. Using mutation-resolved single-cell transcriptomics, we distinguished TET2-mutant and wild-type monocytes within the same aged individuals and identified enrichment of phagocytosis and complement programs in mutant cells. TET2 silencing in human monocytes and macrophages recapitulated this phenotype and enhanced β-amyloid uptake, indicating mutation-specific bias of innate immunity toward aggregate clearance.

Motivation Throughout the years, single-cell RNA sequencing (scRNA-seq) has become a standard approach for characterising transcriptomic changes associated with diseases and other biological conditions. However, the rapid expansion of tools and algorithms developed in various programming languages has made single-cell data analysis increasingly complex. In particular, integrating multiple tools into a single workflow often demands substantial learning time and coding expertise.Results To address these challenges, we developed DoTools, a unified framework for R/Bioconductor and Python/PyPI that simplifies the integration of third-party tools such as scVI, CellTypist, and CellBender into standard pipelines like Seurat, SingleCellExperiment and Scanpy. DoTools provides advanced cross-language wrappers and visualisation utilities to streamline data preprocessing, quality control, cell type annotation, and downstream analysis, while implementing best practices in scRNA-seq analysis regardless of the computational language. Its modular design and compatibility with widely used bioinformatics environments makes it accessible and valuable to both novice and experienced data scientists.Availability and implementation DoTools is freely available for R and Python at Bioconductor and PyPI (https://bioconductor.org/packages/release/bioc/html/DOtools.html and https://pypi.org/project/DoTools-py/), the developmental versions of DoTools are maintained on GitHub (https://github.com/MarianoRuzJurado/DoTools and https://github.com/davidrm-bio/DoTools_py).

Aims Patients with cardiovascular disease (CVD) have an increased risk of developing severe respiratory infections, including COVID-19. However, the underlying molecular mechanisms are not completely understood. It has been previously shown that CVD predisposes to an altered responsiveness to subsequent inflammatory triggers by an imprinted epigenetic memory in innate immune cells. Therefore, we hypothesized that patients with pre-existing atherosclerotic cardiovascular disease (ASCVD) and COVID-19 display a dysregulated inflammatory response compared to patients without ASCVD due to epigenetically altered immune cells leading to increased disease severity. Methods and results Single-cell RNA sequencing revealed a dysregulated myeloid immune response with hyperinflammatory and immunosuppressive features in patients with ASCVD and moderate COVID-19. Assay for Transposase-Accessible Chromatin sequencing and in vitro experiments with isolated monocytes infected with SARS-CoV-2 showed epigenetic priming of monocytes from patients with ASCVD towards increased expression of inflammatory mediators and type I interferon signalling. In a German nationwide cohort (NAPKON), using multiplex cytokine assays, enzyme-linked immunosorbent assays, and bulk-RNA sequencing, we confirmed that patients with ASCVD display an exaggerated inflammatory response during moderate COVID-19. Conclusion This study demonstrates that patients with ASCVD show a dysregulated myeloid immune response in moderate COVID-19 disease. Mechanistically, epigenetic imprinting sensitizes myeloid cells of patients with ASCVD to an exaggerated type I interferon-associated immune response.

AIMS:Cardiovascular disease is the leading cause of death in the European Union and ageing is one of its major risk factors resulting in the progressive deterioration of the cardiac structures and function. Here, we have combined single-nucleus RNA sequencing, imaging, and molecular and cell biology approaches to explore the maladaptive signals that drive cardiac ageing. METHODS AND RESULTS:Single-nucleus RNA sequencing analysis of young (3 months) and old (18 months) murine hearts revealed that the expression of decorin, a secreted proteoglycan expressed in the extracellular matrix of endothelial cells, is induced by ageing. Decorin treatment via osmotic mini pump induced diastolic dysfunction and a pro-inflammatory environment in the myocardium characterized by increased infiltration of immune cells, increased expression of IL-1β in endothelial cells and microvascular leakage in 3-month-old mice. In vitro, decorin treatment induces cardiomyocyte hypertrophy, the expression of different pro-inflammatory cytokines like IL1B in endothelial cells in a TLR2-dependent mechanism, and compromises the endothelial barrier function. CONCLUSIONS:Together, our results identify non-glycanated decorin as a novel player contributing to cardiac ageing and disease. This form of decorin contributes to the age-related structural and functional dysfunction of the heart by inducing a pro-inflammatory environment in the myocardial microvasculature, a hallmark of cardiac ageing.

Aims: Cardiovascular disease is the leading cause of death in the European Union and aging is one of its major risk factors resulting in the progressive deterioration of the cardiac structures and function. Here, we have combined single-nucleus-RNA-sequencing, imaging, and molecular and cell biology approaches to explore the maladaptive signals that drive cardiac ageing. Methods and results: Single-nucleus-RNA-sequencing analysis of young (3 months) and old (18 months) murine hearts revealed that the expression of decorin, a secreted proteoglycan expressed in the extracellular matrix of endothelial cells, is induced by ageing. Decorin treatment via osmotic mini-pump induced diastolic dysfunction and a pro-inflammatory environment in the myocardium characterized by increased infiltration of immune cells, increased expression of IL-1β in endothelial cells and microvascular leakage in 3 months old mice. In vitro, decorin treatment induces cardiomyocyte hypertrophy, the expression of different pro-inflammatory cytokines like IL1B in endothelial cells, and compromises the endothelial barrier function. Conclusions: Together, our results identify decorin as a novel player contributing to cardiac aging and disease. Decorin contributes to the age-related structural and functional dysfunction of the heart by inducing a pro-inflammatory environment in the myocardial microvasculature, a hallmark of cardiac ageing. Translational perspective: Ageing is a major risk factor of cardiovascular disease and the molecular and cellular mechanisms that drive this process have not been completely described. The data presented here identifies decorin as a novel player contributing to systemic inflammation and microvascular dysfunction, two hallmarks of ageing. Although, because of its role regulating TGF-β signalling, decorin has been proposed for anti-fibrotic therapies, the pro-inflammatory effects observed on the cardiac microvasculature should be taken into account for the employment of decorin as an antifibrotic agent to treat disease associated cardiac fibrosis. ### Competing Interest Statement The authors have declared no competing interest.

Background Aging is a major, yet unmodifiable risk factor for cardiovascular diseases, leading to vascular alterations, increased cardiac fibrosis, and inflammation, all of which contribute to impaired cardiac function. However, the microenvironment inciting age-related alterations withing the multicellular architecture of the cardiac tissue is unknown. Methods We investigated local microenvironments in aged mice hearts applying an integrative approach combining single-nucleus RNA sequencing and spatial transcriptomics in 12-week-old and 18-month-old mice. We defined distinct cardiac niches and studied changes in their cellular composition and functional characteristics. Results Integration of spatial transcriptomics data across young and aged hearts allowed us to identify 11 cardiac niches, which were characterized by distinct cellular composition and functional signatures. Aging did not alter the overall proportions of cardiac niches but leads to distinct regional changes, particularly in the left ventricle. Whereas cardiomyocyte-enriched niches show disrupted circadian clock gene expression, vascular niches showed major changes in pro-inflammatory and pro-fibrotic signatures and altered cellular composition. We particularly identified larger vessel-associated cellular niches as key hotspots for activated fibroblasts and macrophages in aged hearts, with interactions of both cell types through the C3:C3ar1 axis. These niches were also enriched in senescence cells exhibiting high expression of immune evasion mechanisms that may impair senescent cell clearance. Conclusion Our findings indicate that the microenvironment around the vasculature is particularly susceptible to age-related changes and serves as a primary site for inflammation-driven aging, so called “inflammaging”. This study provides new insights into how aging reshapes cardiac cellular architecture, highlighting vessel-associated niches as potential therapeutic targets for age-related cardiac dysfunction. ### Competing Interest Statement The authors have declared no competing interest. * ### Nonstandard abbreviations and acronyms CapEC : capillary endothelial cells CE : communication event CM : cardiomyocytes DGE : differential gene expression EC : endothelial cells LVi : left ventricle inner LVm : left ventricle middle LVo : left ventricle outer MP : Macrophages UMI : unique molecular identifiers RV : right ventricle scRNAseq : single-cell RNA-sequencing snRNAseq : single-nucleus RNA-sequencing SEP : septum SMC : smooth muscle cells

Background and aim Aging significantly increases the risk of cardiovascular diseases, characterized by progressive cardiac dysfunction. The vascular niche is crucial for maintaining cardiac homeostasis, yet endothelial cell (EC) impairment during aging remains poorly understood. This study investigates epigenetically regulated mechanisms mediating EC-dependent cardiac aging and identifies a critical role of Zinc finger and BTB domain-containing protein 16 (ZBTB16). Methods Chromatin accessibility (snATAC-seq) and transcriptomic (snRNA-seq) analyses were performed on aged hearts to identify age-related regulatory changes. Functional studies using genetic models, assessed cardiac aging phenotypes. In vitro assays examined EC senescence and secretory profiles, while co-culture experiments analyzed the impact of ZBTB16-deficient EC supernatants on fibroblasts, cardiomyocytes, and neurons. Overexpression experiments in vitro and in vivo tested the potential for ZBTB16 to mitigate aging-associated dysfunction. Results Aged hearts exhibited decreased chromatin accessibility and expression of the transcription factor ZBTB16 in both human and mice. Loss of ZBTB16 in young mice, including Zbtb16 haploinsufficient and endothelial-specific knockout mice, led to premature aging, diastolic dysfunction, and increased secretion of pro-fibrotic and inflammatory factors. Supernatants from ZBTB16-deficient ECs activated fibroblasts, induced cardiomyocyte hypertrophy, and impaired neuronal sprouting. Overexpression of ZBTB16 reversed these effects in senescent ECs and aged mice and reduced diastolic dysfunction. Mechanistic studies identified key downstream targets of ZBTB16, including nuclear receptor-interacting protein 1 (NRIP1). ZBTB16 suppressed NRIP1 expression, limiting fibroblast activation and pro-fibrotic signaling. Conclusions ZBTB16 is a key regulator of endothelial function, maintaining vascular niche homeostasis and mitigating aging-associated cardiac dysfunction. Its loss promotes EC senescence and pro-fibrotic signaling, contributing to diastolic dysfunction. Overexpression of ZBTB16 presents a potential therapeutic strategy for preserving cardiac function during aging. These findings establish a novel role for ZBTB16 in endothelial aging and cardiovascular disease prevention. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. German Research Foundation - DFG, 456687919, 394046768, 390649896 European Research Council, https://ror.org/0472cxd90, 101053352 Dr. Rolf M. Schwiete Stiftung, 2021-033 Frankfurter Forschungsförderung (Goethe University), 71000685 Federal Ministry for Education and Research, 03ZU1202XX German Center for Cardiovascular Research, B22-022 Deutsche Jose Carreras Leukämie-Stiftung, DJCLS 11 R/2020, DJCLS 15 R/2023 Hessian LOEWE Funding Program, III L 5 − 519/03/03.001 – [0015], III 5.7 - 519/03/10.001-(0004) [1]: pending:yes

Chronic heart failure (HF) is characterized by adverse remodeling and persistent inflammation, contributing to impaired heart function and poor prognosis. While the acute immune response post-myocardial infarction (MI) is well-studied, its role in chronic HF remains unclear. Phenotyping of peripheral blood T cells by flow cytometry and single-cell RNA sequencing revealed T central memory cells (TCM) decline, while CD4 + Th17 and CD8 + T effector memory cells increase in HF patients compared to healthy age-matched controls. Furthermore, this decline in TCM cells and increase in homing marker CCR5 on T cell subsets associates with poor prognosis in HF. T cell receptor sequencing revealed clonal expansion in circulating and cardiac T cells, while epitope prediction modeling suggested autoreactivity of T cells in HF. Spatial and scRNA-seq data confirm inflammatory T cell infiltration in the human and murine heart post-MI. In summary, HF shows potential autoreactivity, with an increased homing capacity and declining TCM cells associated with poor prognosis.

BACKGROUND:Aging is a major, yet unmodifiable, cardiovascular risk factor and is associated with vascular alterations, increased cardiac fibrosis, and inflammation, all of which contribute to impaired cardiac function. However, the microenvironment inciting age-related alterations within the multicellular architecture of the cardiac tissue is unknown. METHODS:We investigated local microenvironments in aged mice hearts by applying an integrative approach combining single-nucleus RNA sequencing and spatial transcriptomics of 3- and 18-month-old mice. We defined distinct cardiac niches and studied changes in their cellular composition and functional characteristics. We treated mice with broad-spectrum senolytics dasatinib and quercetin, and endothelial-specific senolytic fisetin and studied their effects on senescence and macrophage populations. RESULTS:Integration of spatial transcriptomics data across 3- and 18-month-old hearts allowed the identification of 11 cardiac niches, which were characterized by distinct cellular composition and functional signatures. Aging did not alter the overall proportions of cardiac niches but led to distinct regional changes, particularly in the left ventricle. While cardiomyocyte-enriched niches showed disrupted circadian clock gene expression, vascular niches showed major changes in proinflammatory and profibrotic signatures and altered cellular composition. We particularly identified larger vessel-associated cellular niches as key hotspots for activated fibroblasts and bone marrow-derived Lyve1- (lymphatic vessel endothelial hyaluronan receptor 1) and resident Lyve1+ macrophages in aged hearts, with interactions of both cell types through the C3:C3ar1 (complement C3 and Complement C3a receptor 1) axis. These niches were also enriched in senescent cells exhibiting high expression of immune evasion mechanisms that may impair senescent cell clearance. Removal of senescent cells by senolytics reduced the presence of Lyve1- macrophages. CONCLUSIONS:Our findings indicate that the perivascular microenvironment is particularly susceptible to age-related changes and serves as a primary site for inflammation-driven aging, so-called inflammaging. This study provides new insights into how aging reshapes cardiac cellular architecture, highlighting vessel-associated niches as potential therapeutic targets for age-related cardiac dysfunction.

Clonal haematopoiesis (CH) is recognized as a potent independent risk factor for cardiovascular disease (CVD). While mutations in common CH-associated genes, such as DNMT3A and TET2 , have been extensively studied, the pathological roles of other CH mutations remain poorly understood. Among these is KDM6A (UTX), an X-linked histone demethylase recently found to be commonly mutated in patients with heart failure. The mechanistic implications of KDM6A mutations in cardiac dysfunction remain largely unknown. Here, using multi-omics profiling and functional characterisation of murine models and patient-derived data, we demonstrate that haematopoietic loss of KDM6A substantially impairs cardiac recovery following myocardial infarction (MI). KDM6A deficiency enhances systemic and cardiac inflammation, characterized by augmented myeloid cell infiltration into the infarcted murine heart. Single-cell chromatin accessibility and single-cell RNA sequencing analyses revealed profound epigenetic and transcriptional reprogramming in KDM6A-deficient myeloid cells, notably CCR2⁺ recruited macrophages and neutrophils. These cells exhibited heightened inflammatory ( Il1b , Nlpr3 , Saa3 ) and chemotactic signatures ( Ccr2 , Mif , Cxcl12 ), increased activation of inflammatory transcription factor networks (AP-1, C/EBP), disrupted chromatin architecture, and enhanced glycolytic activity. Clinically, patients with heart failure harbouring KDM6A-driven CH exhibited increased pro-inflammatory monocyte signatures ( CCR2 , NLPR3 , NFKB1 , FOS , JUN , IL6R , IL32 ), underscoring the translational relevance. Integrative analyses further predicted pathogenic crosstalk between KDM6A-mutated monocytes and cardiac resident cells and was experimentally validated by demonstrating that KDM6A-silenced macrophages drive cardiomyocyte hypertrophy and cardiac fibroblast activation. Our findings establish a critical mechanistic link between KDM6A-driven CH, immune dysregulation, and worsened cardiac outcomes post-MI, highlighting novel avenues for personalized therapeutic strategies in heart failure.

BACKGROUND:Endothelial cells (ECs) play pivotal roles in maintaining cardiac blood supply and regulating inflammation by acting as gatekeepers for immune cell activity. This study unveils a novel immunomodulatory function of cardiac ECs following myocardial infarction. METHODS:We used single-cell RNA sequencing and spatial transcriptomics to identify EC states after acute myocardial infarction in mice. Subsequently, we mimicked the cytokine environment that was predicted to induce EC activation in cell culture studies and confirmed the results in an endothelial-specific deletion mouse model. RESULTS:Single-cell RNA sequencing analysis identified a transient myeloid CD45+CD11b+Cdh5+ immunomodulatory EC phenotype (IMEC) emerging between days 1 and 3 after myocardial infarction. IMECs derived from Cdh5+ tissue resident cells as shown by bone marrow transplantation and lineage tracing experiment. Ligand-receptor interaction predictions indicated a cytokine-mediated activation of IMECs, which we validated through in vitro experiments in cultured ECs. Notably, while cytokine treatment with IL-1β and TGF-β (transforming growth factor β) induced mesenchymal gene expression, the addition of IFN-γ (interferon γ) facilitated the transition into the immunomodulatory phenotype. IMECs exhibited an upregulation of MHC-II (major histocompatibility complex class II) genes, along with the expression of RUNX1 (runt-related transcription factor-1) and proinflammatory cytokines, such as IL-6 and IL-12. IMECs induced T-cell activation through paracrine signaling and were colocalized with T cells in vivo. Inhibition of endothelial-specific IFN-γ-signaling in mice by IFN-γ receptor 1 deletion improved the recovery after myocardial infarction. CONCLUSIONS:These findings provide insight into the role of ECs regulating adaptive immune responses following myocardial infarction, offering potential insights into therapeutic interventions for postinfarction immunomodulation.

Single-cell transcriptomics offers critical insights into the molecular mechanisms of heart failure (HF) with reduced or preserved ejection fraction. However, understanding these mechanisms is hindered by the growing complexity of single-cell data and the difficulty in unmasking meaningful differential gene signatures among HF types. Machine learning, particularly deep neural networks (NNs), address these challenges by learning transcriptional patterns, reconstructing expression profiles and effectively classifying cells but often lacks interpretability. Recent advances in explainable AI (XAI) offer tools to clarify model decisions. Yet pinpointing differentially regulated genes with these tools remains challenging. We introduce a novel method to identify differentially explained genes (DXGs) based on importance scores derived from custom-built NNs. We highlight the superiority of DXGs in identifying HF subtypes-specific pathways that provide new insights into different types of HF. Offering a robust foundation for future research and therapeutic exploration in expanding transcriptome atlases.

Clonal hematopoiesis (CH) due to Tet methylcytosine dioxygenase 2 (TET2) driver mutations is associated with coronary heart disease and a worse prognosis for patients with aortic valve stenosis (AVS). However, it is unknown what role CH plays in the pathogenesis of AVS. In a meta-analysis of All of Us, BioVU, and the UK Biobank, patients with clonal hematopoiesis of indeterminate potential (CHIP) had an increased risk of AVS, with a higher risk among patients with TET2 or ASXL1 mutations. Single-cell RNA-Seq of immune cells from patients with AVS harboring TET2 CH driver mutations revealed monocytes with heightened proinflammatory signatures and increased expression of procalcific paracrine signaling factors, most notably oncostatin M (OSM). Secreted factors from TET2-silenced macrophages increased in vitro calcium deposition by mesenchymal cells, which was ablated by OSM silencing. Atherosclerosis-prone low-density lipoprotein receptor-deficient (Ldlr-/-) mice receiving CH-mimicking Tet2-/- bone marrow transplants displayed greater calcium deposition in aortic valves. Together, these results demonstrate that monocytes with CH promote aortic valve calcification and that patients with CH are at increased risk of AVS.

Heart failure with preserved ejection fraction (HFpEF) accounts for half of heart failure cases and is characterised by reduced pericyte coverage. While the contributions of other cardiac cell types to HFpEF are well-studied, the role of pericytes remains less understood. Using murine single-nucleus RNA-sequencing to study cardiac pericytes in HFpEF, we identified reduced STAT3 expression as a hallmark of HFpEF pericytes. Mechanistic studies in vitro revealed that STAT3 deletion induces cellular senescence and impairs pericyte adhesion, recapitulating HFpEF-like characteristics. These findings suggest that STAT3 is crucial for maintaining pericyte homeostasis and highlight its reduction as a potential driver of pericyte loss, a defining feature of HFpEF.

Aims Members of the VEGF family are crucial modulators of vascular and neural function. While VEGFA signalling has been shown to mitigate several aging-related cardiac phenotypes and prolong survival in aged mice, the role of VEGFB in cardiac aging remains underexplored. In this study, we identify a significant decline in Vegfb expression, particularly of its soluble isoform Vegfb186, in aged mouse and human hearts. To assess the therapeutic potential of VEGFB in aging-associated cardiac pathologies, we used AAV9-mediated gene transfer to overexpress Vegfb186 in 18-month-old male C57Bl/6J mice.Methods and results VEGFB is known to exhibit vascular and neuroprotective effects that we assessed in the ageing heart. In the aged heart, doses of Vegfb186 overexpression that had only a modest effect on the vascular endothelium prevented age-induced diastolic dysfunction and fibrosis. Vegfb186 treatment additionally restored sympathetic and sensory nerve fibre density and increased heart rate variability. Although Vegfb186 overexpression induced cardiac hypertrophy, our findings indicated that this hypertrophy was compensatory rather than pathological as Vegfb186 overexpression corrected the elevated cardiomyocyte length-to-width ratio observed in aged hearts, a metric typically indicative of pathological remodelling. Cardiac single-nucleus RNA sequencing of the hearts and in vitro analysis of the cardiomyocytes indicated up-regulation of the STAT3 signal transduction pathway as a potential contributor of VEGFB-induced cardiac hypertrophy.Conclusion Our findings demonstrate that Vegfb186 overexpression partially reverses age-related cardiac pathologies such as diastolic dysfunction and fibrosis. This work highlights VEGFB as a potential therapeutic target for combating cardiac aging and its associated dysfunctions.

BACKGROUND Heart failure with preserved ejection fraction (HFpEF) is a complex and growing condition, representing over half of all heart failure cases. Despite its high morbidity and mortality, its heterogeneity and limited therapeutic options pose significant challenges. Understanding the molecular mechanisms driving HFpEF is essential for the development of new therapies to improve patient outcomes. METHODS We performed single-nucleus RNA sequencing of nuclei obtained from endomyocardial biopsies of six patients with HFpEF. The obtained dataset was integrated with a dataset of 12 healthy human hearts and their transcriptomic differences were analyzed. RESULTS After quality control and integration of the datasets, nine major cardiac cell types were annotated. HFpEF cardiomyocytes were characterized by a reduction in genes associated with aerobic respiration and fatty acid metabolism and showed an upregulation of RHOA/ROCK1 signaling, which was validated using immunofluorescence staining in human HFpEF myocardial sections. Endothelial cells exhibited signs of increased apoptosis, SEMA3 signaling and signs of reduced VEGFA signaling as well as a reactivation of a fetal gene signature. In line with a prominent role of cardiac fibrosis in HFpEF, we observed increased signs of fibroblast activation and proliferation, and reduced signs of IFNɣ signaling in HFpEF which was most pronounced in activated fibroblasts. Treatment of human cardiac fibroblast with rhIFNɣ resulted in decreased collagen contents. Macrophages from HFpEF myocardium showed a pro-inflammatory transcriptomic signature and showed increased expression of MHC-II molecules. This was associated with signs of an increased IFNɣ response. CONCLUSION Our results provide insights into the transcriptional diversity of HFpEF recapitulating structural, functional, and molecular hallmarks of the disease and provide mechanistic insights which might represent therapeutic targets and biomarkers to improve outcome of patients with HFpEF. What is new? What are the clinical implications? ### Competing Interest Statement The authors have declared no competing interest. * HFpEF : Heart Failure with preserved Ejection Fraction SGLT2 : Sodium/glucose cotransporter 2 ATTR : Transthyretin amylyloidosis ECM : Extracellular matrix EMB : Endomyocardial biopsy PCA : Principal component analysis CCA : Canonical correlation analysis UMAP : Uniform manifold approximation and projection UMI : Unique molecular identifier DEG : Differentially expressed genes MAST : Model-based analysis of single-cell transcriptomics snRNA-Seq : single-nucleus VEGF : Vascular endothelial growth factor rRNA : ribosomal RNA DGE : Differential gene expression FB : Fibroblasts IFNɣ : interferon gamma NF-ϰB : nuclear factor ‘kappa-light-chain-enhancer’ of activated B cells MHC-II : Major histocompatibility complex class HLA : Human leukocyte antigen GO : Gene ontology KEGG : Kyoto encyclopedia of genes and genomes CORUM : Comprehensive Resource of Mammalian Protein Complexes EC : Endothelial cells NO : Nitric oxide

Hematopoietic mutations in epigenetic regulators like DNA methyltransferase 3 alpha (DNMT3A), play a pivotal role in driving clonal hematopoiesis of indeterminate potential (CHIP), and are associated with unfavorable outcomes in patients suffering from heart failure (HF). However, the precise interactions between CHIP-mutated cells and other cardiac cell types remain unknown. Here, we identify fibroblasts as potential partners in interactions with CHIP-mutated monocytes. We used combined transcriptomic data derived from peripheral blood mononuclear cells of HF patients, both with and without CHIP, and cardiac tissue. We demonstrate that inactivation of DNMT3A in macrophages intensifies interactions with cardiac fibroblasts and increases cardiac fibrosis. DNMT3A inactivation amplifies the release of heparin-binding epidermal growth factor-like growth factor, thereby facilitating activation of cardiac fibroblasts. These findings identify a potential pathway of DNMT3A CHIP-driver mutations to the initiation and progression of HF and may also provide a compelling basis for the development of innovative anti-fibrotic strategies.

Background: Cardiovascular diseases, including chronic heart failure with reduced ejection fraction (HFrEF), pose a substantial global health burden, emphasizing the need for better understanding of their genetic and immune-related mechanisms. While the immune system, particularly human leukocyte antigens (HLAs), has garnered attention in cardiovascular research, their specific role in HFrEF remains unclear. This study aims to explore the association between HLA alleles and cardiovascular risk in HFrEF, focusing on disease progression, prognosis, and underlying mechanisms. Methods and Results: Ninety-six chronic HFrEF patients underwent four-digit HLA typing analysis in a case-control study. Analysis of HLA alleles revealed the HLA-DR2 family (mostly HLA-DRB5*0101) carried by 14 patients (15.1%). HLA-DR2 showed the highest significant association with mortality. Additional cohort analysis confirmed elevated cardiac injury markers, including Troponin T, in HLA-DR2 carriers, independent of renal function. Multivariate analysis revealed a higher incidence of myocardial infarction in DRB5 carriers, while non-carriers had higher associations with stroke, CKD, and hyperuricemia. Analysis of 3000 plasma proteins from 30,000 UK participants also showed increased inflammatory (TIMD4,CD80) and CV risk associated (IGFBP7) markers were strongly elevated in HLA-DR2 subjects. Mechanistic insights were obtained through CITE-seq analysis of immune cells from HLA-DR2 carriers and non-carriers, revealing enhanced antigen presentation and pro-inflammatory gene expression in carriers. Functional experiments confirmed that silencing HLA-DRB5 in macrophages reduced antigen presentation and T cell activation. HLA-DR2 carriers also exhibited greater T cell activation and polarization, with HLA-DR2 (specifically HLA-DRB5*0101 allele) silencing in dendritic cells diminishing T cell invasion potential into cardiac organoids. Conclusions: This study associates HLA-DR2 alleles, specifically HLA-DRB5*0101, with prognosis in chronic heart failure and myocardial injury, indicating an increased risk for cardiovascular pathology in carriers. These findings underscore the potential of HLA typing for enhancing risk stratification and informing targeted therapies in HFrEF. Further research is warranted to explore targeted treatments for HLA-DRB5 carriers, offering potential avenues for precision medicine in managing heart failure.