Circulation Research

Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome with incompletely understood molecular mechanisms. Histone serotonylation is a recently identified epigenetic modification in which serotonin is covalently conjugated to glutamine 5 of histone H3 in H3K4me3-marked nucleosomes. Here, we investigated the role of transglutaminase 2 (TGM2)-mediated histone serotonylation in HFpEF. In a mouse model of HFpEF induced by salty drinking water, unilateral nephrectomy and aldosterone infusion (SAUNA), cardiac H3K4me3Q5ser and nuclear TGM2 levels were increased. Cardiomyocyte-specific TGM2-deficient mice developed aggravated HFpEF phenotypes after SAUNA exposure, including worsened diastolic dysfunction, reduced exercise capacity, pulmonary congestion and delayed cardiomyocyte relaxation. CUT&RUN sequencing identified H3K4me3Q5ser-enriched regions predominantly around transcription start sites after SAUNA exposure, with notable enrichment at genes associated with G2/M checkpoint-related stress-response signaling. RNA sequencing further showed that activation of this pathway was impaired in SAUNA-exposed TGM2-deficient hearts. In cardiac myocytes, calcium-binding sites and nuclear localization of TGM2 support checkpoint-related stress-response gene activation in cardiac myocytes. Pharmacological WEE1 inhibition, which activates downstream CDK1-associated checkpoint signaling, partially rescued the aggravated HFpEF phenotype in TGM2-deficient mice. Finally, in patients with HFpEF, lower circulating serotonin levels were associated with adverse cardiac outcomes, and cardiomyocyte H3K4me3Q5ser levels correlated with serum serotonin concentrations. These findings suggest that cardiomyocyte TGM2-mediated histone serotonylation represents a stress-adaptive, cardioprotective epigenetic mechanism in HFpEF.

Background. Myocardial infarction (MI) triggers splenic immune cell trafficking to the heart. Vehicles that carry these signals and mediate this crosstalk are unknown. Hypothesis: We hypothesize that extracellular vesicles (EVs) released post-MI mediate splenic immune trafficking to the heart. Methods. Mice were treated daily with an EV biogenesis inhibitor (GW4869) or vehicle. Splenic/cardiac immune cells were assessed at 3d while survival, cardiac function, hypertrophy, and fibrosis were evaluated at 8w post-MI. Plasma EVs from 1d MI mice or from the hearts that underwent MI/sham in a Langendorff system induced splenic immune trafficking to the heart within 3d and systolic dysfunction at 8w in naive mice. Results. GW4869 i) inhibited splenic regression, ii) increased splenic retention of neutrophils, monocytes, dendritic cells (DCs), and CD4+ T-cells, iii) decreased cardiac gene expression of pro-inflammatory cytokines/chemokines, and iv) decreased trafficking of immune cells to the hearts at 3d post-MI, and iii) improved systolic function and attenuated hypertrophy at 8w post-MI. MI EVs accumulated in the spleen and promoted egress of matured splenic immune cells upon administration to naive mice. Cardiac pro-inflammatory cytokines/chemokines expression and CCR2+MHC-IIhi infiltrating macrophages, CD11c+ DCs, and CD4+ and CD4+TNF+ T-cell levels were also increased in naive mice at 3d post-injection. Importantly, transfer of MI EVs for 2 days induced systolic dysfunction, cellular hypertrophy, and fibrosis in naive mice at 8 w post-injection. DCs process MI EVs for T-cells activation. Conclusions: EVs mobilize splenic immune cells to the heart post-MI and their inhibition can subdue inflammatory tissue-damage to promote healing post-MI.

BackgroundCardiovascular disease and cancer are the two leading causes of morbidity and mortality worldwide. Metabolic dysregulation of cancer cells extends beyond the tumor microenvironment and increases the risk for cardiovascular diseases. One common somatic mutation in cancer cells affects isocitrate dehydrogenase (IDH) 1 and 2, which catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate in the cytosol and mitochondria, respectively. IDH1 and 2 mutations cause the production of the oncometabolite D-2-hydroxyglutarate (D2-HG), which allosterically inhibits -ketoglutarate dehydrogenase (-KGDH) and is associated with reduced cardiac contractile function. MethodsWe combined stable isotope tracer studies with computational modeling to investigate the fundamental role of IDH isoforms in cardiac adaptation under oncometabolic stress. ResultsWe uncovered an unexpected cardiac phenotype that expands the role of IDH1 in the heart beyond oxidative metabolism. We quantified the stable isotopomer distributions from glucose and glutamine in perfused working rat hearts and isolated adult ventricular cardiomyocytes using mass spectrometry-based metabolomics. Our analysis revealed that defective mitochondrial metabolism causes the redirection of carbon flux from oxidative towards reductive pathways. Reductive carboxylation of -KGDH increases glutamine uptake and glutamine-derived citrate formation in working rat heart perfusions and cultured adult mouse ventricular cardiomyocytes. To identify which IDH isoform is responsible for redirecting carbon flux, we developed knockout models of IDH1, IDH2, and IDH3 in adult mouse ventricular cardiomyocytes. Loss of IDH1 expression impaired the reductive formation of citrate and caused functional defects in cardiomyocytes. Lastly, epigenetic analyses of histone marks revealed that IDH1 induces widespread alterations in histone acetylation and tri-methylation. ConclusionOur results highlight a novel role for IDH1 in cardiac metabolism and transcriptional control of metabolic adaptation to tumor-mediated stress and provide evidence that reductive-citrate formation may induce epigenetic modifications in the heart.

Hypertension represents the most prevalent chronic cardiovascular condition, typically culminating in pathological cardiac remodeling characterized by hypertrophy and extensive fibrosis. Although the cellular phenotypes associated with these changes are well-documented, the precise mechanisms by which hypertensive stress is sensed and transduced into a fibrotic program remain poorly defined. To elucidate these mechanisms, we investigated the role of Lysyl oxidase (LOX), an extracellular matrix (ECM)-modifying enzyme that is upregulated during hypertensive stress and associated with cardiovascular diseases. By employing cell-type specific Cre-Lox technology to conditionally delete Lysyl oxidase in either smooth muscle cells (SMCs) or fibroblasts, the primary ECM-secreting cell populations, we demonstrate that fibroblast-specific Lox deletion had no significant impact on the progression of cardiac fibrosis. Conversely, SMC-specific Lox deletion selectively inhibited the fibrotic response without affecting other remodeling parameters, such as cardiac hypertrophy. Notably, in the SMC-specific Lox knockout hearts, fibrosis was restricted to the perivascular niche and failed to propagate into the cardiac interstitium. We find that this transition is a mechanical, ECM-dependent process initiated by SMCs. Our results identify SMCs, rather than fibroblasts, as the primary sensors and initiators of the hypertensive fibrotic response. These findings demonstrate that fibrosis can be uncoupled from other hypertensive manifestations and identify SMC-mediated ECM modification as a potential therapeutic target for treating hypertensive heart disease.

Heart failure with preserved ejection fraction (HFpEF) commonly coexists with atrial fibrillation (AF), but shared mechanisms remain unclear. In this study, we hypothesized that Pitx2, a transcription factor located near the strongest genetic locus associated with AF in humans, increases susceptibility to HFpEF-like remodeling. We also sought to understand pathways that might be central to this increased risk. Male and female Pitx2+/- mice and wild-type littermates received 3-week subcutaneous osmotic pump infusion of saline or angiotensin II (Ang II; 500 ng/kg/min). Cardiac structure and function were assessed by echocardiography and catheterization, and functional capacity by exercise treadmill. RNA transcriptomic profiling was performed to identify candidate pathways. In a separate cohort, Ang II-treated mice were randomized to oral GKT136901 (30 mg/kg/day) or vehicle during infusion. After Ang II infusion, Pitx2+/- mice developed exaggerated HFpEF-like changes, including greater left ventricular hypertrophy, left atrial enlargement, diastolic dysfunction, elevated left ventricular end-diastolic pressure, and reduced treadmill performance. RNA-seq showed enrichment of metabolic and stress-response pathways with selective upregulation of Nox4, confirmed by RT-qPCR. GKT136901 attenuated structural remodeling, diastolic dysfunction indices, elevated filling pressures, and cardiomyocyte hypertrophy, but did not improve endurance. These findings implicate redox signaling, including Nox4, in AF genetic susceptibility-HFpEF interactions.

RationaleSCN1B encodes the {beta}-subunits of the main cardiac voltage-gated sodium channel, NaV1.5. Variants are linked to cardiac conduction disease, often with concealed phenotypes. Whether {beta}1-subunits regulate conduction through nanoscale intercalated disc (ID) structures, e.g. perinexi, and ephaptic coupling remains unresolved. ObjectiveTest whether Scn1b haploinsufficiency induces latent conduction abnormalities that are unmasked by perturbations in extracellular nanodomains. Methods and ResultsAdult Scn1b+/- mice and wild-type (WT) littermates underwent multiscale phenotyping (qRT-PCR, Western blot, patch clamp, transmission electron microscopy (TEM), ex vivo optical mapping, in vivo ECG). Scn1b+/- hearts showed [~]50% reductions in Scn1b mRNA and {beta}1 protein without changes in canonical conduction proteins. Peak sodium current, baseline conduction velocity ex vivo, and baseline QRS duration in vivo were unchanged. However, TEM revealed increased baseline perinexal width in Scn1b+/- hearts. Osmotic expansion of the perinexus with mannitol slowed conduction to a greater extent in Scn1b+/- hearts and prolonged QRS duration in vivo. In contrast, perinexal narrowing with dextran 2MDa selectively increased conduction velocity in Scn1b+/- hearts. ConclusionsScn1b haploinsufficiency preserves baseline excitability and conduction but structurally remodels the ID at the nanoscale, increasing sensitivity to extracellular nanodomain perturbations. These data support a structural role for {beta}1-subunits in ephaptic coupling, and that conduction is maintained over a range of perinexal widths with pathological conduction slowing occurring beyond a critical width. Importantly, osmotic stress unmasks a concealed conduction phenotype, identifying extracellular nanodomain stability as a potential therapeutic target to mitigate arrhythmia risk in SCN1B-associated disease.

BackgroundThis study aimed to investigate whether combined PD-1/CTLA-4 immune checkpoint inhibition predisposes the heart to a hyperinflammatory state, thereby exacerbating cardiac injury following acute myocardial infarction (MI), a critical unresolved question in cardio-oncology. MethodsMyocardial infarction was induced in Pd1-/-Ctla4+/- mice, a genetic model mimicking combined checkpoint inhibition. Key mechanistic insights were gained through in vivodepletion of CD8+ T cells (using anti-CD8a antibody) and pharmacological inhibition of the JAK-STAT1 pathway (using Tofacitinib). Cardiac function, structural injury, and immune responses were comprehensively assessed via echocardiography, flow cytometry, immunofluorescence, and molecular analyses. ResultsCompared to wild-type controls, Pd1-/-Ctla4+/- mice exhibited significantly increased post-MI mortality, worse cardiac function, and larger infarct size. Mechanistically, the aggravated injury was driven by an amplified infiltration of activated, IFN-{gamma}-producing CD8+ T cells, which activated the JAK-STAT1 pathway in macrophages, polarizing them towards a pro-inflammatory state. Depleting CD8+ T cells or inhibiting the JAK-STAT1 pathway effectively attenuated macrophage-driven inflammation and improved all aspects of post-MI injury. ConclusionsCombined PD-1/CTLA-4 blockade exacerbates post-infarction cardiac injury by promoting CD8+ T cell-mediated activation of macrophages via the JAK-STAT1 axis. This work elucidates MI as a context-dependent immune-related adverse event in ICI therapy and identifies CD8+ T cells and the JAK-STAT1 pathway as promising therapeutic targets for cardioprotection in these patients. RESEARCH PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABSO_LIThis study identifies acute myocardial infarction (MI) as a potential, context-dependent immune-related adverse event in the setting of combined PD-1/CTLA-4 checkpoint inhibition, shifting the paradigm beyond the classic focus on myocarditis. C_LIO_LIIt elucidates a novel pathogenic axis where combined checkpoint deficiency exacerbates post-MI injury specifically through CD8+ T cell-derived IFN-{gamma}, which activates macrophages via the JAK-STAT1 pathway. C_LI What Question Should Be Addressed Next?O_LIFuture studies should employ anti-PD-1/CTLA-4 monoclonal antibodies in wild-type or humanized mouse models to validate findings and better recapitulate the pharmacokinetics of clinical ICI therapy, strengthening translational relevance. C_LIO_LIThe long-term consequences of this primed inflammatory state on chronic cardiac remodeling, heart failure development, and the potential interplay with atherosclerosis warrant further investigation. C_LI

Background: Hypoxia develops within growing atherosclerotic lesions, inducing nuclear translocation of hypoxia-inducible factor-1 (HIF1) and metabolic reprogramming. Its role in plaque macrophages and endothelial cells has been studied, but the hypoxic plaque interior is dominated by smooth muscle cell (SMC)-derived cells, for which the role of hypoxia signaling remains unclear. Here, we investigated how loss of Hif1a in SMC lineage cells impacts plaque progression and cell phenotype in murine atherosclerosis. Methods: Atherosclerosis was induced in mice with inducible SMC-specific deletion of Hif1a (Hif1aSMC-KO) and lineage tracing of SMC-derived plaque cells. Plaque size, necrotic core size, calcification, and SMC-derived cell phenotypes were quantified in aortic root sections and gene expression changes mapped by single-cell RNA sequencing. In parallel, a cultured SMC line with or without siRNA-mediated Hif1a knockdown was exposed to hypoxia for assessments of mitochondrial function and reactive oxygen species production. Results: Hif1aSMC-KO mice developed larger plaques, with expanded necrotic cores and increased calcification, compared with littermate controls. SMC-derived plaque cells were more abundant with a higher fraction of Col2a1+ chondromyocytes, and showed elevated markers of proliferation and apoptosis, whereas macrophage and endothelial cell numbers were unaffected. Single-cell RNA sequencing analysis revealed strong dysregulation of mitochondrial genes, including electron transport chain transcripts, along with upregulation of protein folding, proteasome, and oxidative stress response pathways. In cultured SMCs subjected to hypoxia, Hif1a silencing increased cell counts, aggravated mitochondrial proton leak, and led to the accumulation of depolarized, reactive oxygen species-generating mitochondria. Further analysis of SMC-derived cells in plaques from Hif1aSMC-KO mice confirmed increased oxidative stress by 8OHdG staining. Conclusions: HIF1 maintains mitochondrial function and restrains oxidative stress in SMC-derived plaque cells in murine atherosclerosis. Its chronic loss destabilizes redox homeostasis and promotes maladaptive SMC responses, leading to SMC-driven plaque expansion, necrosis, and calcification.

BK channels, coded by the Kcnma1 gene, integrate voltage and intracellular Ca2+ signals and are recognized for their roles in smooth muscle and neuronal excitability. However, their contribution to baseline cardiac physiology remains poorly defined. Here we uncover a fundamental function for BK channels in maintaining normal cardiac performance, independent of pathological stress. Using non-invasive echocardiography, transcriptional profiling, and mechanistic analyses, we demonstrate that Kcnma1 deletion disrupts ventricular function, and remodels metabolic and stress-response pathways. Transcriptomic profiling revealed selective downregulation of mitochondrial uncoupling proteins (UCPs) and suppression of the PGC-1/FOXO3a axis, without broad loss of oxidative phosphorylation components. Enhancing UCP expression restored cardiac performance, indicating that mitochondrial uncoupling and redox control constitute key downstream effectors of BK signaling. Together, these results identify a physiological role for BK channels in maintaining myocardial function and define a mitochondrial BK-UCP axis, critical for cardiac homeostasis.

Metabolic reprogramming is a hallmark of myocardial infarction (MI), in which cardiomyocytes shift from fatty acid oxidation to anaerobic glycolysis, leading to elevated lactate production and mitochondrial dysfunction. Lactylation, a recently described lysine post-translational modification, has emerged as a metabolic signaling mechanism; however, its role within mitochondria during MI remains poorly understood. Here, we define the mitochondrial lactylome following MI and examine how modulation of lactate transport influences mitochondrial metabolism and redox homeostasis. Using quantitative proteomics, we identify extensive remodeling of mitochondrial protein lactylation after MI, affecting enzymes involved in bioenergetics, redox regulation, and metabolic control. Pharmacological inhibition of monocarboxylate transporter-1 (MCT1) using AZD3965 further reshapes the mitochondrial lactylome, increasing lactylation of specific metabolic and redox-associated proteins without uniformly exacerbating mitochondrial dysfunction. Despite sustained impairment of global cardiac function, MCT1 inhibition attenuates post-MI fibrosis and inflammation and partially restores mitochondrial respiratory capacity. Consistent with in vivo findings, genetic or pharmacological inhibition of MCT1 in hypoxic cardiomyocytes-derived cells reduces mitochondrial reactive oxygen species, decreases inhibitory pyruvate dehydrogenase phosphorylation, and improves mitochondrial bioenergetics. Together, these findings reveal that mitochondrial lactylation is a context-dependent regulator of mitochondrial metabolism and redox balance following MI. Rather than acting solely as a pathological modification, lactylation integrates lactate availability with mitochondrial function to influence inflammatory and fibrotic remodeling, highlighting mitochondrial metabolic plasticity as a potential therapeutic target in ischemic heart disease. HighlightsO_LIMyocardial infarction (MI) increases mitochondrial protein lactylation, with 361 identified lactylated proteins. C_LIO_LIAZD3965-mediated MCT1 inhibition further elevates mitochondrial lactylation. C_LIO_LIDistinct alterations in mitochondrial proteins and pathways (TCA cycle, amino acid metabolism, gene expression) were observed. C_LIO_LIAZD3965 reduces cardiac fibrosis and inflammation and partly improves mitochondrial respiration post-MI, but cardiac function remains impaired. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/718938v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@b5a7b3org.highwire.dtl.DTLVardef@14ea92org.highwire.dtl.DTLVardef@1343a29org.highwire.dtl.DTLVardef@1d67716_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

Platelets are increasingly recognized as active regulators of inflammation beyond their canonical hemostatic functions. Although platelets rapidly accumulate in the injured myocardium after myocardial infarction (MI), the mechanisms by which they coordinate the inflammatory response remain poorly understood. Glycoprotein A repetitions predominant (GARP) is a membrane receptor that presents latent transforming growth factor-{beta}1 (TGF-{beta}1) on activated platelets and supports its activation. Given the central role of TGF-{beta}1 in inflammation and tissue repair, we hypothesized that platelet GARP-dependent activation of TGF-{beta}1 regulates inflammatory resolution and repair after MI. Using mice with megakaryocyte- and platelet-specific Garp deletion, we demonstrate that loss of platelet GARP selectively impaired generation of bioactive TGF-{beta}1 without altering platelet reactivity. Following permanent coronary artery ligation, platelet-specific Garp deficiency markedly increased mortality from ventricular rupture and exacerbated adverse left ventricular remodeling, independent of initial infarct size. Transcriptomic and histological analyses revealed heightened endothelial cell activation, increased leukocyte recruitment, delayed inflammatory resolution, and defective extracellular matrix deposition in the absence of platelet GARP. Mechanistically, platelet GARP-dependent TGF-{beta}1 signaling restrained endothelial activation after MI. Together, these findings identify platelet GARP-mediated activation of TGF-{beta}1 as a critical platelet-intrinsic counter-regulatory checkpoint that limits endothelial-driven inflammation and promotes infarct stabilization. Our study reveals an unexpected protective immunoregulatory function of platelets in cardiac repair after ischemic injury.

BackgroundWildland firefighters experience repeated occupational exposure to wildfire smoke at high particulate matter (PM) concentrations, leading to elevated cardiovascular disease risk and hypertension prevalence. However, the pathophysiological processes linking cumulative smoke inhalation to vascular damage and blood pressure elevation remain poorly characterized. To evaluate these effects under controlled exposure conditions, we used a preclinical exposure model calibrated to match the cumulative PM burden deposited in wildland firefighter airways over 7-14 years of service. Male apolipoprotein E knockout (Apoe-/-) mice underwent whole-body inhalation of Douglas fir smoke or filtered air for 2 hours/day, 5 days/week, for 8 or 16 weeks at target PM concentrations of 40 mg/m3. ResultsProlonged smoke exposure induced sustained elevation of circulating tumor necrosis factor-alpha (TNF-), interleukin-1 beta (IL-1{beta}), and interleukin-6 (IL-6), coupled with diffused nuclear factor kappa B (NF-{kappa}B) activation throughout the aortic wall. Smoke inhalation disrupted endothelial adherens junctions, upregulated intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and promoted monocyte recruitment to aortic tissues, concurrent with enhanced monocyte chemoattractant protein-1 (MCP-1) expression. Oxidative stress was evidenced by increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit 2 (NOX2) expression, elevated superoxide levels, and endothelial nitric oxide synthase (eNOS) uncoupling in the aorta, leading to lipid peroxidation and accompanied by intimal apoptosis. These inflammatory and oxidative perturbations occurred alongside a pro-fibrotic phenotypic shift characterized by transforming growth factor beta 1 (TGF-{beta}1) upregulation, myofibroblast differentiation, and progressive collagen accumulation in medial and adventitial compartments of the aortic wall. Functionally, smoke exposure progressively impaired aortic cyclic distensibility through combined wall thickening and circumferential tissue stiffening, while severely attenuating endothelium-dependent and nitric oxide (NO)-mediated vasodilation. These functional and structural shifts culminated in elevated systolic and diastolic blood pressures. While endothelial dysfunction reached maximal impairment by 8 weeks, aortic stiffening continued to worsen through 16 weeks of exposure, demonstrating differential temporal progression of vascular damage. ConclusionsThese findings demonstrate that occupationally relevant wildfire smoke exposure produces convergent inflammatory, oxidative, and profibrotic vascular remodeling with progressive loss of arterial compliance and impaired endothelium-dependent vasodilation, underscoring potential vascular targets for cardiovascular health surveillance and risk mitigation in wildland firefighters.

BackgroundHeart failure with preserved ejection fraction (HFpEF) is a systemic inflammatory syndrome with few effective therapies. Soluble urokinase plasminogen activator receptor (suPAR), a circulating immune-derived glycoprotein, independently predicts adverse outcomes in HFpEF beyond natriuretic peptides, but whether it is a causal driver or a passive marker of inflammatory burden has remained unresolved. MethodsWe tested the hypothesis that elevated circulating suPAR is sufficient to amplify HFpEF by acting on the innate immune system. suPAR-transgenic (suPAR-Tg) and wild-type mice were subjected to a cardiometabolic two-hit model (high-fat diet plus L-NAME) for 15 weeks. Cardiac structure and diastolic function were assessed by serial echocardiography alongside blood pressure, glucose tolerance, and gravimetric endpoints, and left ventricular tissue was profiled by bulk RNA sequencing with in silico cellular deconvolution. Myeloid populations in the heart, spleen, and peripheral blood were quantified by spectral flow cytometry and corroborated by galectin-3 immunofluorescence, and the direct effect of suPAR on macrophages was tested by priming bone marrow-derived macrophages with recombinant suPAR before LPS and IFN-{gamma} stimulation. ResultsSustained suPAR elevation worsened the established HFpEF phenotype, producing greater diastolic dysfunction (higher E/e' and E/A ratios) and pulmonary congestion without altering blood pressure or ejection fraction, indicating a mechanism downstream of the canonical hemodynamic stimulus. Bulk RNA sequencing of left ventricular tissue revealed a coordinated transcriptional shift, with suppression of mitochondrial oxidative phosphorylation and amplification of innate and adaptive immune programs, including interleukin-1{beta} production, leukocyte chemotaxis, and antigen presentation. Spectral flow cytometry demonstrated stepwise expansion of CCR2 inflammatory monocytes and macrophages across cardiac, splenic, and peripheral compartments, corroborated in situ by increased galectin-3 macrophage density. In vitro, recombinant suPAR was not a stand-alone inflammatory ligand but instead primed bone marrow-derived macrophages to markedly amplify TNF-, IL-1{beta}, IL-6, and NLRP3 responses to LPS and IFN-{gamma}. ConclusionsTogether, these findings establish that elevated suPAR is sufficient to act as an upstream amplifier of HFpEF, identify the CCR2 inflammatory monocyte-macrophage axis as its proximate effector, and convert two decades of epidemiologic association into a mechanistically grounded, therapeutically tractable hypothesis with immediate relevance to clinical-stage anti-suPAR antibodies.

BackgroundMetabolic dysfunction-associated steatohepatitis (MASH) is emerging as a risk factor of cardiometabolic diseases, including the atrial fibrillation (AF) - the most common sustained arrhythmia. Given that the liver is a major source of inflammatory mediators, lipids, and hepatokines under metabolic stress, we hypothesized that hepatocyte-derived factors in MASH may accelerate atrial remodeling and arrhythmogenesis. MethodsAnalysis of the Atherosclerosis Risk in Communities (ARIC) visit 5 cohort was performed to determine the association between the FIB-4 index - a classic indicator of liver fibrosis, and AF risk, with multivariable adjustment for common comorbidities. A murine model of MASH was induced using the GAN (Gubra-Amylin NASH) diet. Programmed intracardiac stimulation and echocardiography were performed to assess AF susceptibility and cardiac function. Calcium imaging, histology, flow cytometry, plasma proteomics, and single-nucleus RNA sequencing (snRNA-seq) analyses were employed to elucidate the role of recruited inflammatory macrophages via hepatocyte-derived osteopontin (OPN) in MASH-induced atrial remodeling. ResultsAnalysis of the ARIC cohort confirmed a higher cumulative incidence of AF and an elevated adjusted hazard ratio (HR) in patients with intermediate and high FIB-4 indices compared to individuals with low FIB-4 scores. MASH mice exhibited increased susceptibility to pacing-induced AF, accompanied by enhanced proarrhythmic calcium release events, atrial enlargement, and fibrosis, independent of ventricular dysfunction. Proteomics and snRNA-seq revealed that the hepatocyte-secreted OPN under MASH conditions promoted the differentiation and recruitment of TGFBR1+ inflammatory macrophages to the atria, leading to gasdermin D (GSDMD) activation - an effector of inflammasome signaling and consequent proarrhythmic atrial remodeling. Activation of the monocyte-derived pro-inflammatory TGFBR1+ macrophages was dependent on the OPN receptor CD44. Furthermore, the MASH-induced atrial fibroinflammatory milieu and enhanced AF susceptibility were mitigated through several strategies, including hepatocyte-specific Spp1 (encoding OPN) deletion, neutralization of circulating OPN, ablation of CD44 or GSDMD. ConclusionsThese findings establish a pathogenic role of the hepatokine osteopontin in driving activation and recruitment of TGFBR1+ inflammatory macrophages into the atria, leading to proarrhythmic atrial remodeling under MASH. Osteopontin-targeted therapy or GSDMD inhibition prevents AF, indicating a novel therapeutic strategy for liver disease-related atrial arrhythmogenesis. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIIn the ARIC cohort, metabolic dysfunction-associated steatohepatitis (MASH) is associated with increased risk of atrial fibrillation (AF) after adjusting for common comorbidities. Elevated levels of circulating osteopontin (encoded by SPP1) predict an increased risk of AF in patients with MASH-induced liver fibrosis. C_LIO_LIMASH enhances hepatocyte secretion of osteopontin, leading to expansion of myeloid cells and recruitment of inflammatory macrophages into atria. This liver-to-atrial inflammatory circuit promotes the development of a substrate conducive to AF, which can be attenuated by hepatocyte-specific Spp1 deletion or neutralizing anti-anti-osteopontin antibody treatment to eliminate the mediator, or ablation of inflammasome effector gasdermin D to correct the atrial response. C_LI What are the clinical implications?O_LIOsteopontin may serve as a biomarker for AF in MASH cohorts. C_LIO_LIAnti-osteopontin therapy through neutralizing antibodies may serve as a novel therapeutic strategy for liver disease-related atrial arrhythmia. C_LI

Activation of the immune system impacts the progression of heart failure (HF), but the underlying mechanisms remain incompletely understood. Here, we identify a cardio-intestinal innate immune axis that links systemic venous congestion to myocardial inflammation, fibrosis, and functional decline. Using single-cell and single-nucleus transcriptomic profiling in patients and mice with tricuspid regurgitation (TR), we demonstrate that TR disrupts intestinal barrier integrity and elicits expansion of circulating monocytes which in turn orchestrate pathological crosstalk between the right and left heart. Monocyte-derived Interleukin-6 (IL-6) emerged as a key mediator of TR-driven myocardial fibrosis and dysfunction. Blockade of IL-6 attenuated cardiac fibrosis and improved cardiac function. In patients, catheter-based repair of TR resulted in reduced IL-6 levels. Together, these findings establish cardio-intestinal innate immunity as a mechanism linking altered hemodynamics to left ventricular remodeling and nominate TR patients as a selective target population for IL-6-directed therapy in HF. One Sentence SummaryThis work mechanistically resolves the heart-gut axis in tricuspid valve regurgitation, and its impact on heart failure progression as mediated by Interleukin-6.

BACKGROUND AND PURPOSE{beta}-adrenergic receptors (AR) regulate both cardiac function and remodeling. Many studies suggest that, in addition to their effects on heart rate and contractility, {beta}1-ARs mediate cardiotoxic signaling, whereas {beta}2-ARs are generally cardioprotective. However, there is conflicting data on the role of {beta}2-ARs, differing dependent on the nature of the stress. Given the extremely common use of {beta}-blockers and agonists clinically, we sought to understand the differential cardioprotective/cardiotoxic effects of {beta}2-AR signaling dependent on timing (acute vs. chronic) and type of cardiotoxic stress. EXPERIMENTAL APPROACHWild-type (WT) and {beta}-AR knockout ({beta}1-KO and {beta}2-KO) mice were subjected to acute (15 mg{middle dot}kg-1 x 1 dose) or chronic (2 mg{middle dot}kg-1{middle dot}wk-1 x 7 wks) oxidative stress using doxorubicin (DOX). Survival, cardiac function and histopathology were assessed and differential signaling activation determined by Western blot and gene expression by RNA-seq. KEY RESULTSWe have shown that {beta}2-KOs manifest extreme cardiotoxicity with acute DOX (100% mortality within 30 min), supporting a strong cardioprotective role of {beta}2-signaling. In marked contrast, with chronic DOX, {beta}2-KO had enhanced survival (t[1/2] 54 d vs. 42 d in WT) and attenuated cardiac dysfunction. In {beta}2-KO, acute DOX activated stress MAPKs (p38, ERK and JNK), whereas chronic DOX did not; furthermore, in the absence of {beta}2-ARs, oxidative stress and lipid accumulation were reduced, genes regulating compensatory metabolic pathways (AMPK and insulin/PI3K) were upregulated, and genes regulating mitochondrial and contractile function were preserved, whereas they were downregulated in WT with chronic DOX. CONCLUSIONS{beta}2-AR signaling switches from being cardioprotective during acute oxidative stress, to cardiotoxic during chronic stress. Inhibition of {beta}2-AR signaling during chronic stress induces signaling and metabolic compensations that serve to reduce oxidative injury. This unexpected temporal switching has potential significant implications for all models of cardiovascular disease, as well as for the clinical use of subtype-specific {beta}-blockers. CLINICAL PERSPECTIVEO_ST_ABSWhat is new?C_ST_ABSO_LIOur finding that {beta}2-adrenergic receptor signaling can switch from being beneficial (cardioprotective) to detrimental (cardiotoxic) depending on the acuteness or chronicity of a cardiac stressor. C_LIO_LIIdentification of the mechanisms by which this temporal switch is mediated could lead to new drug development. C_LI What are the clinical implications?O_LIOur findings provide potential guidance in choosing between a {beta}1-specific vs. a {beta}1/2-non-specific drug when treating specific cardiovascular diseases based on their temporal characteristics. C_LIO_LIThe temporal protective/toxic switching that we describe could be a mechanism common to many other drugs, yet is rarely tested, suggesting the need for additional studies using temporal course as a factor. C_LI

BackgroundImpaired myocardial metabolism is a defining feature of heart failure, but many defective pathways and mechanisms remain to be identified. Prior studies find phosphoglycerate kinase and its synthesized product 3-phospho-glycerate required for the serine synthetic pathway (SSP) are reduced in human HFpEF myocardium. As serine is also provided exogenously, the impact of SSP reduction is uncertain. Here, we tested if and how SSP decline coupled to phosphoglycerate dehydrogenase (PHGDH) impacts cardiomyocyte (CM) and whole heart metabolic remodeling and stress responses. MethodsStudies were performed in isolated CMs and mice with CM-selective knock-down of PHGDH. Using pharmacological inhibition or genetic silencing of PHGDH, we tested their impact on CM one-carbon metabolism pathways, cell hypertrophic responses, mitochondrial respiration, and in vivo functional, structural, and metabolic adaptations to pressure-overload stress. ResultsIn CMs, PHGDH inhibition caused dose-dependent serine depletion linearly coupled with cytotoxicity, accompanied by NAD/NADH and GSH/GSSG imbalance, reduced ATP, and disruption of one-carbon and nucleotide metabolites. Stable-isotope tracing revealed distinct metabolic fates of glucose-derived (SSP) versus exogenous serine. Exogenous serine did not rescue PHGDH-deficient CMs, whereas combined ribose and an anti-oxidant (DTT) attenuated injury and reduced nucleotide pools. PHGDH suppression reduced amino acid abundance, impaired nascent protein synthesis, and blunted endothelin-1-induced hypertrophic and mitochondrial respiration. In vivo, cardiomyocyte-specific PHGDH heterozygous mice (PHGDH+/-) had no basal phenotype, but amplified chamber dilation, dysfunction, fibrosis, and mortality 4 weeks after transverse aortic constriction (TAC). Corresponding increases in amino acids, one-carbon metabolites, nucleotides, and TCA-cycle intermediates in wild-type TAC hearts were significantly blunted in PHGDH+/- hearts. ConclusionsCardiomyocyte SSP is a critical regulator of redox balance, one-carbon metabolism, purine synthesis, amino acid homeostasis, and growth-related pathways required for cardiac adaptation to pressure overload. It is non-redundant with exogenous serine by providing distinct influences on key metabolic pathways and is a potential therapeutic target.

BackgroundCalcific aortic valve stenosis (AVS) is the most prevalent valvular heart disease in Western adults, yet no disease-modifying therapy exists. High shear stress (HSS) generated by progressive valvular obstruction drives endothelial injury and immune-mediated inflammation, but the contribution of circulating T cells to AVS pathogenesis remains poorly defined. ObjectivesWe tested whether chronic HSS corresponds with epigenomic reprogramming of peripheral T cells proportionate with hemodynamic severity to yield a clinically informative proxy of disease. MethodsA prospective cohort of 70 participants was recruited for peripheral blood sampling, including 34 with severe symptomatic AVS (aortic valve area <1.0 cm2, mean gradient [&ge;]40 mmHg) scheduled for transcatheter aortic valve implantation and 36 age- and sex-matched controls. Peripheral T cells were isolated and profiled by genome-wide CpG methylation (Illumina MethylationEPIC) and RNA-sequencing. To test whether HSS directly activates inflammatory signaling, Jurkat T cells were exposed to 20 dyn/cm2 HSS via parallel-plate microfluidic chamber and concomitant CD3/CD28 stimulation, followed by assessment of NFAT nuclear translocation and NFAT target gene expression. ResultsUnsupervised clustering of the 5,000 most-variable CpG loci resolved an epigenomic axis segregating AVS from control T cells (PC1, 15.8% variance explained; P = 3.9x10-6). Multivariable-adjusted analysis identified 3,950 differentially methylated positions (1,889 hyper-, 2,061 hypo-methylated), enriched in promoter-associated CpG islands implicating aortic valve morphogenesis (P = 6.0 x 10-10) and cell-cell adhesion pathways (P = 9.5 x 10-5). Multi-omics factor analysis isolated a latent factor that independently associated with AVS (adjusted P = 1.8x10-3; AUC = 0.79), enriched for chemokine receptor binding and TNF-family signaling, and correlated with canonical HSS-responsive transcripts, consistent with a T cell-mediated shear stress activation. An 18-CpG elastic-net methylation risk score discriminated AVS from controls (AUC = 0.89) and independently predicted hemodynamic severity ({beta} = 7.05 mmHg/SD, 95% CI 2.31-11.79). HSS augmented NFAT nuclear translocation in CD3/CD28-activated Jurkat T cells and induced NFAT-responsive inflammatory transcripts. ConclusionsSevere AVS is associated with promoter-enriched epigenomic remodeling of circulating T cells that converges on hemodynamic stress-dependent inflammatory programs. An 18-CpG methylation risk score outperforms clinical covariates and tracks hemodynamic severity, establishing peripheral T cell DNA methylation as a molecular corollary of AVS.

BackgroundRare heterozygous loss-of-function (LoF) variants in SVIL, encoding the Z-disk and costameric protein supervillin, have recently been identified as a cause of hypertrophic cardiomyopathy (HCM). Although supervillin is implicated in actin-dependent mechanotransduction, the mechanisms linking SVIL deficiency to cardiomyopathy remain poorly understood. Homozygous LoF cause a novel skeletal Myofibrillar Myopathy-10 (MFM-10) while heterozygous LoF cause HCM without skeletal myopathy. In this study we use a human model system to disentangle the LoF pathomechanism of the scaffolding protein supervillin in cardiomyocytes and its clinical implications. MethodsUsing CRISPR/Cas-9 we engineered a representative pathogenic LoF variant Q255X into an isogenic induced pluripotent stem cell (iPSC) line creating the heterozygous SVILQ255X/+ and homozygous SVILQ255X/Q255X cell lines. These lines were differentiated into iPSC-derived cardiomyocytes (iPSC-CMs) and cellular phenotypes were assessed using bulk RNA-sequencing, LC-MS proteomics, electrophysiological and calcium handling analyses, contractility measurements, sarcomere organization analysis, Seahorse metabolic flux assay, and pharmacological intervention with mavacamten. ResultsThe Q255X variant resulted in SVIL haploinsufficiency at both RNA and protein levels with no evidence of a truncated protein. Compared with isogenic controls, SVILQ255X/+ iPSC-CMs demonstrated action potential shortening, calcium transient elongation, sarcomeric disorganization and hypertrophy, and impaired mitochondrial respiration. Multi-omic analyses of SVILQ255X/+ iPSC-CMs showed a profile of cellular stress and inflammation, hypertrophic and pro-fibrotic signalling, and a pseudohypoxic state driven by decreased respiration and a HIF-induced glycolytic shift. These abnormalities were not present in SVILQ255X/Q255X cardiomyocytes, consistent with a relatively limited cardiac phenotype reported in homozygous variant carriers. Mavacamten improved sarcomeric disorganization and hypertrophy in SVILQ255X/+ cells but did not rescue energetic compromise. ConclusionsPathogenic heterozygous SVIL LoF produces a distinct cellular phenotype characterized by impaired mechanotransduction, mitochondrial dysfunction, and maladaptive metabolic remodelling that promotes hypertrophic and pro-fibrotic signalling. These findings define a mechanistic basis for SVIL-associated cardiomyopathy and identify metabolic dysfunction as a potential therapeutic target beyond sarcomere-directed therapy. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LISVIL haploinsufficiency causes HCM through a mechanism distinct from canonical sarcomeric disease, characterized by impaired mechanotransduction, mitochondrial dysfunction, and pseudohypoxia-driven metabolic remodeling. C_LIO_LIHeterozygous SVIL loss of function produces a substantially more severe cardiomyocyte phenotype than homozygous loss of function, providing a mechanistic explanation for the predominance of cardiac disease in heterozygous variant carriers. C_LIO_LIMavacamten improves sarcomeric organization but does not restore impaired mitochondrial respiration, demonstrating that energetic dysfunction persists despite sarcomere-directed therapy. C_LI What Are the Clinical Implications?O_LIOur findings give functional evidence to support SVIL as a clinically relevant HCM disease gene and its inclusion in clinical genetic testing panels. C_LIO_LIThese findings establish SVIL-associated cardiomyopathy as a mechanistically distinct form of HCM and offer insight into the pathomechanism of Z-disk and costameric HCM C_LIO_LIThe persistence of mitochondrial dysfunction despite myosin inhibition suggests that drugs targeting mitochondrial bioenergetics may be a therapeutic strategy in patients with SVIL-associated cardiomyopathy. C_LI

IntroductionAortic stenosis (AS) is characterised by progressive aortic valve (AV) leaflet fibrosis and calcification, yet no medical therapies exist to slow disease progression. AV interstitial cells (VICs) that differentiate into myofibroblasts are central drivers of fibrosis. The Ca2+-activated K+ channel KCa3.1 promotes pro-fibrotic signalling in several fibrotic diseases, however its role in AS remains unknown. MethodsKCa3.1 protein expression was examined in paraffin embedded tissue by Immunohistochemistry from control and AS valve tissue. VICs were isolated, cultured and phenotypically characterised as myofibroblasts from AV tissue obtained from patients with severe tricuspid AS undergoing surgical AV replacement (n=19). KCa3.1 mRNA and protein expression were assessed by qRT-PCR and immunohistochemistry, and functional channel activity confirmed using patch-clamp electrophysiology. The effects of transforming growth factor-{beta}1 (TGF{beta}1) stimulation and pharmacological inhibition with the selective KCa3.1 blocker senicapoc were examined. ResultsImmunoreactive KCa3.1 channels and smooth muscle actin were detected in both control and AS aortic valve tissue, localised to elongated, nucleated interstitial cells, with significantly higher expression observed in AS tissue compared to control. Isolated VICs exhibited an activated myofibroblast phenotype, expressing THY-1, vimentin, collagen and -smooth muscle actin (SMA) (n=9). Myofibroblasts expressed KCa3.1 mRNA and protein and demonstrated functional plasma membrane channels. TGF{beta}1 stimulation increased KCa3.1, SMA and collagen type I mRNA expression, while KCa3.1 blockade with senicapoc (100 nM) significantly attenuated TGF{beta}1-induced SMA expression, stress fibre formation and collagen gel contraction. Senicapoc had no effect on myofibroblast proliferation or migration. ConclusionsWe show for the first time that functional KCa3.1 channels are expressed in human AS tissue and AV myofibroblasts, where they regulate myofibroblast contraction, -SMA expression, and differentiation, promoting pro-fibrotic activity. These responses are attenuated by the selective KCa3.1 inhibitor senicapoc. Given its established safety in phase 3 clinical trials, KCa3.1 inhibition represents a promising and readily translatable anti-fibrotic therapeutic strategy for AS.