Circulation Research

RationaleHypertrophic cardiomyopathy (HCM) is a common inherited cardiac disorder characterized by cardiac hypertrophy, fibrosis, arrhythmias, and sudden cardiac death (SCD). Although current therapies primarily target sarcomere dysfunction, the contribution of epigenetic dysregulation to HCM pathogenesis and its therapeutic potential remain poorly understood. ObjectiveTo determine whether pharmacological inhibition of histone lysine demethylases (KDMs) with JIB-04 can prevent or reverse HCM progression and to identify the underlying epigenetic mechanisms. Methods and ResultsWe evaluated the pan-KDM inhibitor JIB-04 in Myh6R403Q/+ mice carrying the murine equivalent of the pathogenic human MYH7 R403Q mutation. JIB-04 prevented disease progression, reduced cardiac hypertrophy and fibrosis, preserved cardiac function, and completely prevented SCD in cyclosporin A- accelerated HCM. JIB-04 also reversed established disease, produced sustained therapeutic benefits after drug withdrawal, and improved cardiac function in aged mice with spontaneous HCM. Bulk RNA sequencing and ATAC-seq demonstrated partial restoration of disease-associated transcriptional programs and chromatin accessibility. Proteomic analyses identified PHF2 (KDM7C) as a candidate target of JIB-04 in both mouse and human HCM hearts. PHF2 knockdown suppressed hypertrophic, inflammatory, and fibrotic gene expression in cardiomyocytes, macrophages, and fibroblasts, respectively. Human HCM hearts exhibited increased expression of multiple JIB-04-sensitive KDMs, including PHF2. In MYH7 R403Q induced pluripotent stem cell- derived cardiomyocytes, JIB-04 normalized disease-associated gene expression, restored connexin-43 membrane localization, and improved mitochondrial respiration. Although prolonged treatment induced reversible hepatomegaly with hepatic lipid accumulation, co-administration of the antioxidant N-acetylcysteine mitigated liver toxicity while preserving the therapeutic efficacy of JIB-04. ConclusionsPharmacological KDM inhibition prevents and reverses HCM through epigenetic remodeling of disease-associated transcriptional and chromatin programs. These findings identify KDM inhibition as a promising therapeutic strategy for HCM, establish PHF2 as a candidate mediator of disease pathogenesis, and support further development of KDM-targeted therapies.

Controversy surrounds the lineage potential of cardiac cells, particularly epicardial cells, during heart development, largely due to the non-specific expression of epicardial marker genes and the resulting non-specific labeling in Cre-loxP mouse models. Using DARLIN mice, a CRISPR/Cas9-based lineage-tracing system independent of the Cre-loxP system, we analyzed the lineage development of embryonic cardiac cells in an unbiased manner and identified lineages shared among different cell types, such as epicardial cells and vascular endothelial cells (Vas_ECs). To further confirm the lineage potential of epicardial cells, we identified an epicardial cell-specific marker gene, Lrrn4, through analysis of a multi-staged single-cell mRNA-sequencing (scRNA-seq) dataset, and generated a corresponding Lrrn4-CreER mouse line. We then bred this line with a reporter mouse to confirm its specificity for labeling epicardial cells, and subsequently performed prolonged lineage tracing, which revealed specification of the labeled epicardial cells into Vas_ECs. Finally, Using this mouse line, we investigated epicardial cell function by selectively ablating these cells and by expressing TGF{beta} in epicardial cells to convert their lineage from Vas_ECs to fibroblasts. Both approaches resulted in significant developmental defects in embryonic hearts. Together, these results indicate that epicardial cells can give rise to Vas_ECs, and that the Lrrn4-CreER mouse model is a valuable tool for elucidating the role of the epicardium in heart development.

Background: Cardiac fibrosis is a hallmark of many cardiovascular diseases, driven by sustained fibroblast activation and excessive extracellular matrix deposition, leading to myocardial stiffening and impaired contractility. Current therapies inadequately address this process. This study evaluated the antifibrotic potential of lanifibranor, a balanced pan-peroxisome proliferator-activated receptors (PPARs) agonist, in TGF-beta1-induced cardiac fibrosis. Methods: Human cardiac microtissues, along with 2D and 3D cardiac fibroblast and cardiomyocyte cultures, were used to assess cell viability, structure, metabolism, contractility, and gene expression. Results: Lanifibranor reduced TGF-beta1-induced fibrosis by limiting fibroblast activation and matrix deposition without affecting viability. In fibroblasts, these effects were associated with partial restoration of mitochondrial respiration and reduced focal adhesion maturation. In cardiac microtissues, lanifibranor improved contraction kinetics, decreased profibrotic transcriptional activity, and preserved bioenergetic homeostasis despite altered nucleotide balance. In cardiomyocytes, treatment normalized contractility and calcium handling while maintaining metabolic stability. Conclusions: Lanifibranor attenuates TGF-beta1-driven cardiac fibrosis by combining antifibrotic effects with metabolic and functional improvements in human models.

Fatty acid oxidation (FAO) is a major energy source in the adult heart, and disruption of cardiac metabolism is closely associated with heart failure. Here, we investigated the effects of cardiac-specific overexpression of the H19X-encoded miR-424(322)/-503 cluster using an inducible transgenic mouse model. Sustained miR-424(322)/-503 overexpression caused rapid metabolic and functional deterioration, with early impairment of fatty acid oxidation. Short-term induction reduced FAO activity and downregulated genes involved in lipid metabolism, while glycolytic enzyme activity remained largely unchanged. Continued miR-424(322)/-503 expression subsequently led to severe dilated cardiomyopathy characterized by ventricular dilation, wall thinning, fibrosis, reduced contractility, and high mortality. Importantly, disease progression was dependent on the level and duration of miR-424(322)/-503 expression, as intermittent or lower-dose induction delayed cardiac dysfunction and prolonged survival. Withdrawal of miR-424(322)/-503 expression after the onset of dysfunction promoted substantial functional recovery. Together, these findings identify miR-424(322)/-503 as a potent regulator of cardiac metabolic reprogramming that disrupts fatty acid metabolism and drives progressive heart failure.

Myocardial infarction is a leading cause of death and disability worldwide. Ischemic injury leads to irreversible loss of cardiomyocytes, the contractile cells of the heart, and formation of a fibrotic scar. After infarction, macrophages massively infiltrate the heart and orchestrate the tissue repair process by removing dead cells and modulating fibroblast activation for scar formation. We previously demonstrated that diverse monocyte-derived macrophage populations dynamically accumulate in the heart following myocardial infarction, notably a pro-repair Trem2hi subset. In this study, we leveraged spatial transcriptomics, single-cell RNA-seq, and functional assays to elucidate the role of TREM2 in driving macrophage-mediated cardiac tissue repair post-infarction. We show that Trem2hi macrophages localize in scarring areas of the infarcted myocardium in the vicinity of collagen-producing myofibroblasts. In Trem2-/- mice, cardiac accumulation of monocyte-derived macrophages with a pro-scarring matrisome-associated macrophage signature was reduced. TREM2 deficiency was functionally associated with reduced fibroblast proliferation, accumulation of myofibroblasts, decreased collagen deposition in the infarcted heart, and increased infarct size. In vitro, we show that TREM2 mediates efferocytosis-induced pro-fibrotic gene expression and promotes macrophage ability to induce fibroblast migration. IL-4 priming of bone marrow-derived macrophages further increased the pro-fibrotic response in macrophages, suggesting that IL-4 and efferocytosis act synergistically to drive this phenotype. Altogether, our results show that TREM2 is essential for the accumulation and function of pro-scarring monocyte-derived macrophages in the infarcted myocardium.

BackgroundDuchenne muscular dystrophy (DMD) cardiomyopathy is characterized by progressive microtubule remodeling, connexin-43 (Cx43) dysregulation, and ventricular arrhythmias. We previously demonstrated phospho-mimic knock-in of {beta}III-tubulin S172E preserves microtubule organization and attenuates cardiac pathology in mdx mice. However, whether these protective effects can be reproduced using a clinically relevant gene-delivery strategy remains unknown. Methods and ResultsWe generated a cardiomyocyte-specific adeno-associated virus serotype 9 (AAV9) vector expressing phospho-mimic {beta}III-tubulin (Tubb3-S172E) under the cardiac troponin T promoter and delivered it to 4-5-month-old wild-type and mdx mice. Cardiac Tubb3-S172E expression was confirmed by quantitative qPCR and immunoblotting. In mdx mice, AAV9-mediated Tubb3-S172E expression significantly reduced mononuclear inflammatory infiltration, restored Cx43 localization at intercalated discs, and attenuated isoproterenol-induced arrhythmia susceptibility. In contrast, cardiac fibrosis, Nav1.5 protein expression, and peak sodium current density were not significantly improved. Overexpression of wild-type {beta}III-tubulin in healthy hearts increased Cx43 lateralization and arrhythmia susceptibility, indicating that {beta}III-tubulin phosphorylation state rather than protein abundance determines its protective function. ConclusionsCardiomyocyte-targeted delivery of phospho-mimic {beta}III-tubulin partially recapitulates the protective effects observed in the genetic S172E knock-in model. These findings identify {beta}III-tubulin Ser172 phosphorylation as a critical regulator of microtubule-dependent electrical remodeling and support therapeutic modulation of this pathway in Duchenne muscular dystrophy cardiomyopathy. Research PerspectiveO_LICardiomyocyte-targeted AAV9 delivery of phospho-mimic aIII-tubulin improves Cx43 organization, inflammatory remodeling, and arrhythmia susceptibility in dystrophic hearts, demonstrating that therapeutic modulation of {beta}III-tubulin Ser172 phosphorylation partially recapitulates the protective effects observed in the genetic S172E model. C_LIO_LIThe dissociation between improved electrical remodeling and persistent Nav1.5 and fibrotic abnormalities suggests that {beta}III-tubulin Ser172 phosphorylation selectively regulates specific microtubule-dependent pathological pathways in dystrophic cardiomyopathy. C_LIO_LIFuture studies should define the molecular mechanisms linking {beta}III-tubulin Ser172 phosphorylation to cardiomyocyte-immune cell communication and determine how this pathway coordinates electrical and inflammatory remodeling in dystrophic hearts. C_LI

Background: HFpEF accounts for roughly half of heart failure admissions and lacks disease-modifying therapy. Autotaxin (ENPP2) generates lysophosphatidic acid (LPA), a profibrotic and pro-inflammatory bioactive lipid. Whether circulating lysophospholipid metabolism is altered in HFpEF, and whether autotaxin inhibition modifies an established experimental HFpEF phenotype, is untested. Methods: Plasma from patients with HFpEF (n=210) and non-heart-failure comparators (n=27) underwent untargeted and LPA-targeted mass spectrometry and a nine-analyte multiplex immunoassay. Male C57BL/6J mice received a high-fat diet plus L-NAME (0.85 g/L) or chow for 5 weeks; after phenotype confirmation, they received oral PF-8380 (30 mg/kg/day) or vehicle for 10 weeks. Endpoints were echocardiography, functional assessment, gravimetric studies, tail-cuff pressure, trichrome fibrosis, and flow cytometry of heart and spleen. Results: All nine analytes, including the autotaxin protein ENPP2, were higher in HFpEF than comparators. HFpEF plasma showed higher LPE O16:1, LPE O18:2, PS 38:4 and PC 36:4;O, and lower SM 39:2; O3 and PS 36:0. LPA 20:0 was 3.5-fold higher in both sexes, whereas LPA 18:2 was lower in women. Diet plus LNAME raised blood pressure, LV mass, and isovolumic relaxation time with preserved ejection fraction. PF-8380 reduced echocardiographic indices of diastolic dysfunction, fibrosis area, cardiomyocyte area, and cardiac CD11b+, CD64+, CD86+, and Ly6G+ frequencies, without altering fat or lean mass. Conclusion: In male mice with established two-hit HFpEF, autotaxin inhibition improved diastolic indices and reduced fibrosis, hypertrophy, and cardiac myeloid accumulation. Human data show altered lysophospholipid composition. Collectively, these findings nominate the autotaxin/LPA axis as a tractable therapeutic target and support further evaluation of autotaxin inhibition as a candidate disease-modifying strategy for HFpEF management.

Background and AimsCardiac fibrosis is a major contributor to heart failure (HF), yet mechanisms limiting pathological fibroblast activation remain incompletely understood. We identified dysferlin (DYSF), a membrane repair protein, as highly induced in HF fibroblasts and investigated its role in regulating profibrotic responses. MethodsCardiac fibroblasts from patients with end-stage HF and unaffected donor hearts were analysed by liquid chromatography-tandem mass spectrometry and bulk RNA sequencing. Dysferlin expression was validated in independent cohorts. Selected gene/protein expression was validated using single-cell/single-nucleus RNA sequencing and multiplex immunofluorescence of human myocardium from dilated cardiomyopathy (DCM), ischaemic cardiomyopathy (ICM), acute myocardial infarction (AMI), and unaffected hearts. Functional studies were performed in human and mouse cardiac fibroblasts using siRNA-mediated silencing and TGF-{beta} stimulation, and in engineered human 3D cardiac microtissues. Fibrotic remodelling, autophagy, apoptosis, and contractile function were assessed by molecular, histological, biochemical and functional analyses. ResultsDysferlin abundance was markedly increased in HF fibroblasts. Across HF myocardium, DYSF was enriched in activated fibroblasts but largely excluded from COMP-enriched fibrotic regions, consistent with a role in restraining fibroblast state transitions. Although induced by TGF-{beta}, DYSF silencing enhanced extracellular matrix production, increased FOSL2 expression, and promoted differentiation into COMP-positive matrifibrocytes. In engineered human cardiac microtissues, DYSF silencing exacerbated fibrosis, increased apoptosis, and impaired contractility. Mechanistically, dysferlin restrained the TGF-{beta}-FOSL2-autophagy signalling axis, whereas FOSL2 suppressed DYSF expression, defining a reciprocal regulatory circuit. Silencing FOSL2 or MXRA5 increased dysferlin levels, while mRNA-protein discordance implicated S-acylation as a potential regulator of dysferlin protein abundance. ConclusionsDysferlin is a stress-inducible antifibrotic regulator that limits maladaptive fibroblast differentiation and myocardial fibrosis, thereby representing a potential therapeutic target to attenuate adverse cardiac remodelling in HF. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/745492v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@b52f7dorg.highwire.dtl.DTLVardef@140f781org.highwire.dtl.DTLVardef@3964f5org.highwire.dtl.DTLVardef@131404_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundAdipose tissue surrounding the heart and vasculature plays critical roles in cardiovascular homeostasis and disease, yet the cellular and molecular milieu of these depots at single-cell resolution remains incompletely characterized. Understanding how regional adipocytes differ transcriptionally and communicate with neighboring cardiovascular cells is essential for developing targeted therapeutic strategies. MethodsWe performed single-nucleus RNA sequencing (snRNA-seq) on human adipose tissue from four anatomically distinct depots: ascending aorta, left atrium, right coronary artery, and subcutaneous fat. We characterized cellular composition, adipocyte and progenitor heterogeneity, depot-specific transcriptional programs, and intercellular communication networks. We further examined signaling remodeling in disease contexts, including atrial fibrillation and aortic aneurysm. ResultsWe identified six transcriptionally distinct adipocyte subpopulations and six adipocyte stromal and progenitor cell (ASPC) subpopulations were shared across depots but showed marked differences in abundance and gene expression reflecting developmental imprinting, including HOX family genes and anterior-posterior patterning programs. Intercellular communication analysis revealed depot-specific ligand-receptor interactions, with EPHA signaling identified as selectively enriched in the left atrial adipose depot. Disease-state analyses demonstrated extensive change in cell-cell communication in atrial fibrillation and aortic aneurysm, with differential regulation of FN1, EGF, SLIT, NOTCH, and CD46 signaling pathways. ConclusionsOur study reveals that cardiac and vascular adipose depots harbor transcriptionally specialized adipocytes and progenitors with distinct intercellular communication programs that are remodeled in atrial fibrillation and aortic aneurysm.

BackgroundHeart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome associated with metabolic stress, hypertension, systemic inflammation, and microvascular dysfunction. Early cell-type-specific events and intercellular communication programs that accompany disease onset and progression remain poorly defined. MethodsWe performed a longitudinal study of HFpEF progression in high-fat diet (HFD)+L-NAME mice at control/baseline (0 weeks, 0w/Ctrl), early (1w), intermediate (4w), and established (8w) stages. Metabolic, hemodynamic, exercise, echocardiographic, and single-cardiomyocyte function were assessed. Cardiac non-cardiomyocytes (non-CMs) were profiled by single-cell RNA sequencing (scRNA-seq), with bulk RNA-seq for tissue-level comparison. Endothelial remodeling was assessed in an L-NAME-independent HFD plus mild transverse aortic constriction model (HFD+mTAC) and a published human HFpEF single-nucleus RNA-seq cohort. An endothelial-macrophage adhesion assay tested whether HFpEF-mimic stress promotes endothelial activation and macrophage adhesion. ResultsIn the HFD+L-NAME model, metabolic dysfunction, hypertension, reduced exercise tolerance, abnormal diastolic filling with preserved ejection fraction, and altered cardiomyocyte calcium handling were detected by 1w and persisted through 8w. Bulk RNA-seq showed progressive remodeling, with limited change between 8w and 12w, guiding scRNA-seq timepoint selection. scRNA-seq of 94,848 cardiac non-CMs identified nine major populations with stage-dependent remodeling. Endothelial cells (ECs) were recovered in high proportion and showed an early, pronounced transcriptional response, with inflammatory, adhesion, interferon-response, migratory, and vascular-remodeling programs emerging by 1w. Related EC activation signatures were observed in HFD+mTAC and human HFpEF data. Functionally, HFpEF-mimic stress increased adhesion and chemokine expression in human ECs and enhanced macrophage adhesion. Fibroblast matrix remodeling occurred at later stages, while macrophages progressively shifted toward inflammatory states. CellChat suggested stage-dependent communication remodeling from early endothelial-immune interactions toward later macrophage-fibroblast crosstalk. ConclusionTime-resolved scRNA-seq reveals coordinated, stage-dependent remodeling of the cardiac microvascular and interstitial microenvironment during HFpEF progression. Early endothelial activation emerges before later fibroblast matrix remodeling and inflammatory macrophage remodeling, identifying candidate cell states and signaling pathways for future mechanistic investigation. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIThis study provides a time-resolved single-cell atlas of the cardiac non-cardiomyocyte compartment across baseline, early, intermediate, and established stages of HFpEF progression, rather than a single late-stage snapshot. C_LIO_LIEndothelial cells exhibit early inflammatory, adhesion, interferon-response, and vascular-remodeling programs within the first week of disease, preceding the later predominance of fibroblast matrix remodeling and inflammatory macrophage remodeling. C_LIO_LIThis endothelial activation signature is supported across two mechanistically distinct HFpEF mouse models and aligns with endothelial inflammatory and vascular-remodeling programs in human HFpEF myocardium, supporting its translational relevance. C_LI What Are the Clinical Implications?O_LIEarly endothelial activation may represent a targetable stage of HFpEF pathogenesis that arises before more established structural and fibrotic remodeling. C_LIO_LITherapeutic strategies aimed at limiting endothelial inflammatory activation or endothelial-immune interactions may help attenuate downstream vascular, immune, and stromal remodeling in HFpEF. C_LIO_LIThese findings provide a preclinical foundation for future longitudinal human studies testing whether early endothelial activation can serve as a biomarker, therapeutic target, or disease-staging feature in HFpEF. C_LI

Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening disease characterized by progressive medial degeneration, impaired mechanical integrity, and extracellular matrix (ECM) degradation. However, no pharmacological therapy has been proven to halt aneurysm progression or prevent dissection or rupture. Vascular smooth muscle cells (VSMCs) are vital for maintaining medial architecture by sensing and remodeling the surrounding ECM; however, the mechanism by which abnormal ECM mechanics are transmitted to nuclear transcriptional programs that disrupt aortic wall matrix homeostasis remains incompletely understood. Integrative transcriptomic screening of Lysyl oxidase (LOX)-deficient and ?-aminopropionitrile (BAPN)-induced TAAD models identified vestigial-like family member 4 (VGLL4) as a mechanosensitive transcriptional regulator of TAAD. VGLL4 was enriched in VSMCs and markedly increased in aortas from patients with TAAD and BAPN-induced TAAD mice. VSMC specific deletion of Vgll4 protected mice from BAPN-induced aortic dilation, dissection, rupture-associated mortality, vascular stiffening, ECM degradation, and medial destruction. Mechanistically, pathological matrix remodeling and mechanical stress induced VGLL4 expression in VSMCs, where VGLL4 cooperated with specificity protein 1 (SP1) to activate Wisp1 transcription. In vivo, VSMC-enriched Wnt-inducible signaling pathway protein (WISP1) overexpression exacerbated TAAD progression, whereas Wisp1 knockdown protected against BAPN-induced TAAD and mitigated the severe aortic phenotype driven by VGLL4 overexpression. Secreted WISP1 bound Tissue Inhibitor of Metalloproteinases 3 (TIMP3) through its C-terminal domain and impaired TIMP3-mediated MMP9 inhibition, thereby increasing MMP9 proteolytic activity and accelerating ECM degradation. Consistently, in vivo Wisp1 knockdown protected against BAPN-induced TAAD. Together, these findings define the VGLL4-WISP1-TIMP3/MMP9 axis, which couples pathological ECM mechanics to nuclear transcriptional activation and protease-dependent matrix degradation in VSMCs. This pathway promotes medial structural failure, aortic mechanical stability loss, and TAAD progression, identifying WISP1 as a potential therapeutic target for preserving aortic wall matrix homeostasis.

Background: Cardiometabolic diseases frequently involve concurrent cardiovascular and hepatic dysfunction, yet the conserved molecular mechanisms underlying these systemic responses remain poorly defined. Objectives: To identify conserved molecular responses across complementary manifestations of cardiometabolic stress and determine whether integrated multi-organ analyses reveal therapeutically actionable targets for heart failure. Methods: Cardiac functional phenotyping, hepatic injury profiling, and bulk RNA sequencing were performed across three complementary mouse models representing distinct manifestations of cardiometabolic stress: high-fat diet plus L-NAME (HFD+LN)-induced heart failure with preserved ejection fraction (HFpEF; cardiovascular disease), Western diet (WD)-induced obesity (systemic metabolic stress), and choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-induced steatotic liver disease (hepatic metabolic stress). Comparative transcriptomic analyses distinguished organ-specific responses from conserved molecular signatures. Results: Each model produced distinct systemic, hepatic, and cardiac phenotypes accompanied by divergent transcriptional responses within individual organs. Cross-model and cross-organ integration identified a limited set of conserved molecular responses to cardiometabolic stress, with Serpine1, encoding plasminogen activator inhibitor-1 (PAI-1), emerging as a highly conserved candidate that exhibited preferential induction in the heart. Pharmacologic inhibition of PAI-1 significantly improved cardiac function and attenuated adverse remodeling in established HFpEF, whereas hepatic pathology was comparatively less affected, indicating differential organ-specific dependence on this pathway. Conclusions: Integrated analyses across complementary manifestations of cardiometabolic stress identified conserved molecular signatures that transcend individual disease models and organs. These findings establish a comparative framework for discovering cardiovascular therapeutic targets and identify PAI-1 as a promising mediator of cardiac remodeling in cardiometabolic disease.

Background: Atrial fibrillation (AF) burden is increasingly recognized as a determinant of clinical risk. Currently, AF burden can only be estimated using long-term rhythm monitoring. Bone morphogenetic protein 10 (BMP10) is a protein secreted from cardiac atria associated with AF and AF-related complications. This study evaluated whether BMP10 concentrations are associated with AF burden in a human atrial model: atrial engineered heart tissue (aEHT). Methods: Human induced pluripotent stem cell-derived atrial cardiomyocytes were cast into atrial engineered heart tissues (aEHTs). To mimic AF burden, mature aEHTs were optogenetically-paced at a high rate of 4 Hz, either intermittently for 4 hours every 2 days (~10% burden) or continuously for 24 hours per day (100% burden). After 18 days of high-rate pacing intervention, 7 days of recovery without pacing followed. BMP10 release was quantified by ELISA and contractile function was assessed by video analysis. EHT transcriptional remodeling in response to mimicked AF burden was assessed by RNA sequencing and the effect of recovery was analyzed by qPCR. Results: High-rate optogenetic pacing mimicking AF lead to a dynamic, burden-dependent BMP10 release: BMP10 concentrations in the medium were increased by intermittent optogenetic pacing (~10% burden) and highest under continuous optogenetic pacing (100% burden). BMP10 release declined toward control levels during recovery. Contractile dysfunction was most impaired after continuous pacing and showed only partial recovery within 7 days after pacing cessation. RNA sequencing revealed distinct burden-dependent transcriptional states. Pacing-regulated transcripts were related to BMP/TGF{beta} signaling, atrial identity, calcium handling, contractile phenotype, and electrophysiological remodeling. After recovery, BMP10 mRNA expression remained elevated despite normalization of BMP10 protein release. Conclusions: AF burden dynamically regulates BMP10 release and functional and molecular remodeling in human aEHTs. BMP10 release depicts a secreted protein-based readout of current or recent atrial high-rate stress, whereas persistent transcriptional changes indicate molecular memory of prior AF burden. These findings support BMP10 release as a burden-sensitive AF biomarker

Introduction: Vascular calcification is a detrimental ageing-related pathology that is markedly accelerated in metabolic disorders. It is driven by osteogenic differentiation of vascular smooth muscle cells (VSMCs), however epigenetic regulatory pathways activated early in this transition remain poorly defined. Methods: An in vitro calcification model was developed using primary human aortic VSMCs cultured with or without mineral stress. Epigenetic changes were assessed using targeted PCR arrays and CUT&RUN sequencing. Key findings were validated in vivo using single-cell sequencing datasets from human large arteries and spatial transcriptomic analysis in atherosclerotic carotid plaques. Transcriptomic and CUT&RUN analyses identified gene targets altered by epigenetic remodelling, and molecular tools were applied to study effects on metabolism, inflammation, apoptosis, and calcification. Results: During early calcification in response to mineral stress, SWI/SNF chromatin remodelling complexes shift toward ncBAF enrichment in pre-osteogenic VSMCs. ncBAF complexes activated transcriptional programs involved in inflammation, apoptosis, and glycolysis-all hallmarks of calcifying VSMCs. The transcription factor ETS2 was identified as a novel component of ncBAF complexes. Disruption of ncBAF or ETS2 impaired osteogenic differentiation and calcification. Notably, ETS2 expression was regulated by ncBAF, forming a positive feedback loop that reinforced VSMC phenotypic switching. Co-activation of ETS2 and ncBAF and the resulting transcriptional shifts were confirmed in human arterial single-cell datasets, with osteogenic/inflammatory clusters showing NFkB and RUNX2 activation. Spatial transcriptomics further suggested that a macrophage-rich microenvironment may promote the differentiation of smooth muscle cells toward an overt osteogenic/inflammatory phenotype. Immunohistochemistry showed that ETS2 levels correlated with calcification severity in human vessels supporting the potential clinical relevance of ETS2. Conclusions: Our findings identify a novel epigenetic mechanism in vascular calcification, where ncBAF and ETS2 cooperate to drive VSMC phenotypic switching. This ncBAF-ETS2 axis represents a potential therapeutic target to modulate VSMC plasticity and intervene early in the progression of cardiovascular calcification.

Mitochondrial metabolism undergoes dramatic reprogramming during postnatal cardiac maturation, yet the enzymatic regulators that ensure continuity of TCA cycle flux in this period remain incompletely defined. FAHD1 is a mitochondrial oxaloacetate decarboxylase (ODx) with proposed roles in modulating the activity of Complex II of the electron transport chain (ETC), but its physiological relevance in vivo has remained unclear. Here, we identify FAHD1 as a critical regulator of mitochondrial function with strong impact on cardiomyocyte (CM) maturation. Using a germline Fahd1-knockout (KO) mouse model, we show that Fahd1 deficiency impairs Complex II respiration, reduces pyruvate levels, and induces a compensatory metabolic shift toward glycolysis and anabolic biosynthesis. Loss of FAHD1 disrupts sarcomere organization, delays the fetal-to-adult myosin isoform switch, and leads to left ventricle systolic dysfunction and cardiomyocyte hypertrophy. These findings highlight FAHD1 as a mitochondrial gatekeeper and potential target for modulating cardiac development and disease.

Transverse (t)-tubules enable rapid, synchronous Ca release required for efficient cardiac contraction by bringing L-type Ca channels into close apposition with ryanodine receptors. In heart failure with reduced ejection fraction (HFrEF), t-tubule disorganisation and loss occur alongside cardiac microtubule remodelling, contributing to impaired Ca handling and contractile dysfunction. Despite their canonical function in contraction, how t-tubules develop is unknown. Microtubules support delivery of L-type Ca channels to t-tubules via Amphiphysin-II/BIN1, yet whether microtubules directly regulate t-tubule formation and maintenance is unclear. Here, we investigated a role for microtubules in t-tubule development and homeostasis. Neonatal rat ventricular myocytes (NRVMs), which lack endogenous t-tubules, were used as a reductionist model in which BIN1 overexpression induces nascent membrane tubules. Microtubule depolymerisation with nocodazole before BIN1 overexpression impaired BIN1-driven tubule formation, reducing tubule density and length. Dynein inhibition with EHNA produced similar effects, indicating a requirement for microtubule-based motor activity during tubule elongation. Knockdown of the microtubule +TIP tracking protein CLIP-170 also reduced BIN1-driven tubule density, implicating BIN1-CLIP-170-dependent microtubule capture in tubule initiation. Microtubules were also required to maintain existing tubules. In NRVMs with established BIN1-driven tubules, microtubule depolymerisation, microtubule stabilisation or dynein inhibition each reduced tubule density and length. Consistent with this, acute microtubule depolymerisation or stabilisation disrupted native t-tubule networks in isolated adult sheep left atrial myocytes. Together, these findings identify cardiac microtubules as active regulators of t-tubule architecture. We propose that BIN1-dependent tubule formation requires CLIP-170-mediated microtubule plus-end capture and dynein-dependent elongation, while ongoing microtubule dynamics are necessary to preserve mature t-tubule structure.

Background and AimsMutations in the desmin (DES) gene cause a variety of cardiomyopathies associated with arrhythmias, yet the electrophysiological consequences of these variants remain largely uncharacterized. The aim of this study was to investigate the pathogenic mechanisms of the de novo DES p.R406W variant, which was identified in a 9-year-old patient who suffered from severe ventricular arrhythmias and sudden cardiac death without overt structural heart disease. MethodsHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the DES p.R406W variant (including the patients line) were compared to isogenic controls. Action potentials (AP) of hiPSC-CMs were recorded using patch-clamp. Furthermore, 3D engineered heart tissues (EHTs) were generated from hiPSC-CMs and their APs were recorded with sharp microelectrodes. Analytical techniques also included transmission electron microscopy (TEM) and integrated transcriptomic and proteomic profiling. Finally, a heterozygous knock-in (KI) mouse model carrying the Des p.R405W ortholog was evaluated through surface ECG, echocardiography and ex vivo cardiac optical mapping. ResultsThe DES p.R406W mutation prolonged AP duration in IM-R406W hiPSC-CMs and EHTs vs Control ones. Multi-omics analysis of EHTs revealed a dysregulation of genes and proteins involved in contractile function, cell adhesion, and electrical activity. TEM imaging revealed changes in Z-disc architecture in mutant tissues. Twenty-week-old Des p.R405W KI mice exhibited ventricular conduction slowing (prolonged QRS) and a high susceptibility to ventricular tachyarrhythmias, likely due to reentrant mechanisms. Mild hypertrophy was also observed, but only in females. ConclusionThe DES p.R406W variant is highly pathogenic, causing electrical and structural remodeling of the myocardium. This study highlights the effectiveness of hiPSC-CMs and EHTs in recapitulating the clinical phenotype of desminopathy, providing a platform for investigating the mechanisms of early-onset cardiac arrhythmias and SCD.

Background: Heart failure (HF) is classified by ejection fraction: reduced EF (<40%) is HFrEF and preserved EF (>50%) is HFpEF. Unlike HFrEF, no therapeutic agent improves mortality in HFpEF. The molecular mechanism that produces HFpEF is not completely understood, but the cascade of pathology that produces HFpEF is thought to begin with changes in vascular reactivity, including a decrease in NO mediated vasodilatation, which coupled with subsequent changes in contractility, energetics and coronary blood flow produce HFpEF. If abnormal vascular reactivity is the initial step in the pathological cascade that produces HFpEF, restoring and/or improving vascular reactivity could represent a novel treatment strategy. Vascular reactivity is primarily regulated by myosin light chain phosphatase, which has catalytic, myosin targeting (MYPT1) and 20kDa subunits. Alternative mRNA splicing of exon24 (E24) of the MYPT1 transcript produces MYPT1 isoforms that differ by the presence or absence of a COOH-terminal leucine zipper (LZ+/LZ-); E24 exclusion produces an NO responsive LZ+ MYPT1, while E24 inclusion produces an NO unresponsive LZ- MYPT. Methods: We used the mouse two-hit model of HFpEF (high fat diet and L-NAME) and treated mice with an antisense octo-guanidine targeting the 5' splice site of E24 (ASO-E24) to increase the expression of the NO responsive, LZ+ MYPT1 isoform in vascular smooth muscle. Invasive and noninvasive hemodynamics were used to determine LV function. Results: Compared to mice with HFpEF, ASO-E24 treatment maintains LZ+ MYPT1 expression (4.7{+/-}0.7au v 1.0{+/-}0.4au v 2.0{+/-}0.4au, control v HFpEF v ASO-E24 Rx, p<0.05), improves diastolic function; LVEDP (10{+/-}1mmHg v 20{+/-}4mmHg v 14{+/-}3mmHg, p<0.05), dP/dtmin (-8000{+/-}300mmHg/s v 6000{+/-}500mmHg/s v 8500{+/-}700mmHg/s, p<0.05), both early (E; 0.60{+/-}0.05m/s v 0.42{+/-}0.06m/s v 0.64{+/-}0.06m/s, p<0.05) and late diastolic filling (A; 0.38{+/-}0.03m/s v 0.24{+/-}0.02m/s v 0.47{+/-}0.04m/s, p<0.050 and also prevents the increase in lung weight (167{+/-}5g v 175{+/-}7g v 166{+/-}5g, p<0.05). Further, mice treated with ASO-E24 maintained normal relaxation to 8Br-cGMP (65{+/-}5% v 44{+/-}9% v 72{+/-}9%, p=0.05). Conclusion: These data demonstrate that maintaining normal LZ+ MYPT1 expression and vascular reactivity prevent the development of HFpEF. These results are consistent with the hypothesis that abnormal vascular reactivity is the initial and primary step in the pathological cascade that produces HFpEF and ASO-E24, which is designed to preserve normal LZ+ MYPT1 expression and vascular reactivity, could represent a novel and effective treatment strategy for HFpEF.

Background: APOE4, the variant of apolipoprotein E carried by 25% of individuals, is a common genetic risk factor for cardiovascular disease (CVD). Although ApoE classically participates in lipid transport, APOE4-associated risk goes beyond impact on circulating lipids. Life-threatening CVD events including myocardial infarction and stroke are driven by atherogenesis and thrombosis. In mice ApoE4 increases atherosclerosis severity, but whether other major drivers of CVD events are influenced by ApoE4 is unknown. Methods: GWAS data for venous thromboembolism (VTE) were analyzed. In humanized APOE3 (hE3) and APOE4 (hE4) mice, thrombosis was assessed by intravital microscopy (IVM) in the mesenteric microcirculation and by inferior vena cava (IVC) partial ligation. Actions of ApoE3 versus ApoE4 on endothelial cells (EC) and their underpinnings were studied in cultured human and mouse aortic EC, interrogating interactomes with immunoprecipitation-mass spectrometry and quantifying the secretion of Von Willebrand Factor (vWF), a critical initiator of thrombosis. Single cell transcriptomics datasets were queried do localize endothelial cell gene expression. Results: GWAS showed that APOE4 is associated with increased VTE risk, and whereas plasma lipids were similar, both microvascular and venous thrombosis were markedly increased in hE4 compared to hE3 mice. In cultured EC, whereas ApoE3 attenuated vWF secretion, it was enhanced by ApoE4, and both processes were mediated by ApoE receptor 2 (ApoER2). ApoE4, but not ApoE3, suppressed VEGF eNOS activation and NO production by causing the recruitment of the protein phosphatase 2A (PP2A) catalytic subunit to ApoER2 and the activation of PP2A. PP2A deletion prevented ApoE4-induced eNOS antagonism and vWF secretion by preserving Akt activation, and the NO donor spermine NONOate negated apoE4 stimulation of vWF secretion. PP2A activity was increased in hE4 aortas and IVC, and EC ApoER2 deletion or pharmacologic PP2A inhibition fully prevented exaggerated thrombosis in hE4 mice. In human great saphenous vein ApoER2 is primarily expressed in valvular endothelium. Conclusions: APOE4 is a risk allele for thrombosis, and ApoE4 is prothrombotic in microvasculature and veins in mice. Mechanistically, the ApoE4-EC ApoER2 tandem enhances vWF secretion by recruiting and activating PP2A and antagonizing eNOS, resulting in exaggerated thrombosis. In human veins ApoER2 is expressed in valvular endothelium, which is the most common site of initiation of venous thrombosis. Targeting these processes may afford protection from both primary thrombotic disorders like VTE and acute CVD events such as myocardial infarction and stroke in 25% of the population.

Chikungunya virus (CHIKV) infection is increasingly linked to cardiovascular complications, but the mechanisms underlying CHIKV-induced cardiovascular disease (CVD) remain unclear, and targeted therapies are lacking. Although elevated interleukin-17A (IL-17A) levels have been reported in CHIKV patients and associated with cardiovascular pathology, its role in CHIKV-induced cardiac disease is poorly defined. To address this question, we employed our newly developed heterozygous interferon /{beta}/{gamma} receptor-deficient (Ifnag+/-) mice and primary human cardiac fibroblasts to investigate the contribution of IL-17A signaling to CHIKV-associated cardiac pathology. We found that CHIKV infection induced IL-17A production in the heart, and that mice deficient in Il17a (Il17a-/-) and in its receptor gene, Il-17ra (Il17ra-/-), exhibited marked resistance to CHIKV infection in both cardiac tissue and primary cardiac fibroblasts. Genetic deletion of IL-17A signaling significantly enhanced type I interferon responses and decreased viral burden in mouse hearts. Interestingly, blockade of IL-17RA with an FDA-approved monoclonal antibody for plaque psoriasis, Brodalumab, drastically increased type I interferon production and reduced viral replication in both human cardiac fibroblasts and human embryonic kidney 293 (HEK 293) cells. In addition, inhibition of IL-17A signaling suppressed the expression of pro-inflammatory mediators, including Il-1{beta}, Tnf-, and Cxcl2, reduced immune cell infiltration into cardiac tissue, and mitigated cardiac injury. Importantly, therapeutic blockade of IL-17A signaling after CHIKV infection reduced viral replication in both the heart and circulation. Collectively, these findings identify IL-17A signaling as a critical regulator of CHIKV replication and cardiac inflammation and highlight the IL-17A/IL-17RA axis as a promising therapeutic target for CHIKV-associated cardiovascular disease. ImportanceChikungunya virus (CHIKV) infection has been frequently associated with cardiovascular complications, yet the host pathways that promote viral infection and cardiac injury remain poorly understood. Here, we identify IL-17A signaling as a previously unrecognized regulator of CHIKV pathogenesis in the heart. Using a novel heterozygous interferon receptor-deficient mouse model and primary human cardiac fibroblasts, we demonstrate that IL-17A signaling facilitates CHIKV replication via suppressing antiviral type I interferon responses. Genetic deletion or pharmacological blockade with an FDA-approved monoclonal antibody of IL-17A signaling reduced viral burden, attenuated inflammatory cytokine production, limited immune cell infiltration, and protected against cardiac injury. Importantly, therapeutic inhibition of IL-17A signaling after infection remained effective in reducing viral replication in both cardiac tissue and circulation and mitigating cardiac damage. These findings reveal a critical role for the IL-17A/IL-17RA axis in linking antiviral immunity to CHIKV-induced cardiovascular disease and identify a potential translatable therapeutic target for CHIKV-caused cardiac complications.