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.

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. 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.

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.

BackgroundPulmonary hypertension (PH) is characterized by pulmonary vascular remodeling and smooth muscle cell accumulation, but the progenitor-like cells that contribute to this process remain incompletely defined. MethodsWe combined analyses of human pulmonary arterial hypertension lungs and experimental PH models with bulk and single-cell RNA sequencing, lineage tracing, inducible ablation of CD133+ cells, and conditional deletion of Cxcr4 in CD133+ cells. ResultsCD133 expression was markedly increased in human and experimental PH lungs. Transcriptomic analyses identified inflammatory, metabolic, chemokine-associated, and smooth muscle cell-like programs in CD133+ cells from PH lungs. Lineage tracing showed that CD133+ cells contributed to endothelial and smooth muscle cell compartments during experimental PH. Genetic ablation of CD133+ cells attenuated hypoxia-induced PH and pulmonary vascular remodeling, whereas Cxcr4 deletion in CD133+ cells reduced PH severity. ConclusionsCD133+ progenitor cells are functional contributors to pulmonary vascular remodeling, and CXCR4 signaling mediates their pathogenic activity. Targeting pathogenic CD133+ cell states or CXCL12/CXCR4 signaling may provide a strategy to limit vascular remodeling in PH.

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.

Impaired contractility and reduced myocardial energetic reserve underlie heart failure with reduced ejection fraction. Catecholaminergic inotropes such as dobutamine are used to augment cardiac output. However, dobutamine increases Ca{superscript 2} cycling, raising ATP demand and worsening energetic stress. The myotrope CK-138 increases contractility by directly activating myosin, sparing the added energetic cost of Ca{superscript 2} handling. This study compares CK-138 and dobutamine with respect to the relationship between contractile performance and myocardial energetic state, including high-energy phosphate balance, energetic efficiency, and substrate-specific metabolic fluxes. Isolated rat hearts were perfused with escalating concentrations of CK-138 or dobutamine. Contractility was assessed by measuring left ventricular pressure and rate-pressure product. Myocardial energetics were analyzed using 31P-NMR, and metabolic fluxes by 13C NMR and mass spectrometry. Unlike dobutamine, CK-138 increased LV contractility without increasing heart rate or LV end-diastolic pressure. CK-138 preserved ATP and phosphocreatine levels, maintaining a stable phosphocreatine-to-ATP ratio and free energy of ATP hydrolysis, whereas dobutamine progressively depleted both. At comparable workload, dobutamine exhibited higher glycolytic flux and lactate production, indicating greater reliance on glycolysis relative to mitochondrial oxidative metabolism, whereas CK-138 exhibited a 13% higher rate of ATP synthesis and [~]50% lower anaplerotic flux, consistent with preserved mitochondrial efficiency. In conclusion, CK-138 enhances cardiac contractility while preserving myocardial energetic state and substrate utilization. Unlike dobutamine, which depletes ATP reserves and shifts metabolism toward glycolysis, CK-138 maintains ATP homeostasis and supports oxidative metabolism. These findings support cardiac myosin activators, including CK-138 and omecamtiv mecarbil, as a mechanistically distinct class of energy-efficient inotropes.

BackgroundNeprilysin (NEP) is a zinc-dependent metalloprotease targeted in heart failure therapy to prevent it degrading circulating cardioprotective vasoactive peptides. NEP can also cleave sarcolipin (SLN), the skeletal- and atrial muscle-specific micropeptide regulator of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). A direct pathophysiological role of NEP in ventricular muscle has not been established. MethodsProteomics and immunoblot analysis of human myocardial specimens were used to quantify NEP abundance in failing and non-failing hearts. Heterologous protein expression and biochemical binding assays assessed NEP-mediated cleavage of phospholamban (PLB) and its impact on PLB-SERCA interactions. Functional consequences of NEP expression or inhibition were evaluated in neonatal rat ventricular myocytes and in a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of heart failure. ResultsWe observed increased NEP abundance in failing human myocardium relative to non-failing controls. We demonstrated that NEP cleaves phospholamban (PLB), disrupting PLB-SERCA interactions. Mutation of PLB (V49A), prevented NEP cleavage and preserved PLB-SERCA binding, indicating V49 is critical for NEP substrate recognition. In neonatal rat ventricular myocytes, NEP expression was associated with faster Ca2+ transient decay kinetics and increased SR Ca2+ load, consistent with reduced SERCA inhibition. Inhibition of NEP in a hiPSC-CM heart failure model attenuated the hypertrophic transcriptional responses and reversed Ca2+-transport dysregulation. ConclusionsThese findings implicate increased NEP expression in the sarcoplasmic reticulum of cardiomyocytes as previously unrecognized maladaptive consequence of heart failure contributing to cardiac dysfunction. In this novel pathophysiological mechanism, increased NEP results in PLB cleavage and loss of regulation of SERCA. While this may relieve SERCA inhibition and augment cellular Ca2+ handling, loss of PLB chronically disrupts hearts dynamic response to adrenergic stress, changing heart rate, or other physiological challenges. The data provide new insight into the cardioprotective effects of pharmacological NEP inhibition in clinical practice, reveal a novel mechanism of action of neprilysin inhibition in cardiomyocytes and may help inform future therapeutic strategies for patients with heart failure. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/732949v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@4daf93org.highwire.dtl.DTLVardef@41ef8aorg.highwire.dtl.DTLVardef@d568daorg.highwire.dtl.DTLVardef@d6d213_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

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

BackgroundPulmonary hypertension (PH) is a progressive vascular disease characterized by endothelial dysfunction, vascular remodeling and increased pulmonary vascular resistance. The {beta}3-adrenergic receptor ({beta}3-AR) has been implicated in cardiovascular regulation and cardioprotective mechanisms; however, its role in pulmonary vascular disease remains poorly understood. We investigated whether activation of {beta}3-AR protects pulmonary endothelial function and prevents the development of pre-capillary PH. Methods{beta}3-AR expression was evaluated in pulmonary endothelium from patients with Chronic Obstructive Pulmonary Disease (COPD) and in murine models of hypoxia-induced PH. Genetic mouse models including {beta}3-AR knockout (KO) and conditional {beta}3-AR overexpression in endothelial cells (EC) or in smooth muscle cells (SMC), were used to determine cell-specific roles. Pharmacological activation of {beta}3-AR was achieved using the selective {beta}3-agonist mirabegron in hypoxia-induced PH mice and monocrotaline-induced PH rats. Pulmonary vascular reactivity and vasodilatory responses to {beta}3-AR stimulation were evaluated by wire myography in isolated pulmonary arteries. Mechanistic studies were performed in human pulmonary artery endothelial cells (HPAEC) under hypoxic conditions, in human pulmonary arterial smooth muscle cells (HPASMC) and in endothelial nitric oxide synthase (NOS3) KO mice. Results{beta}3-AR was upregulated in pulmonary endothelium of COPD patients and mice exposed to chronic hypoxia. Genetic deletion of {beta}3-AR aggravated PH, whereas endothelial-specific overexpression attenuated the disease phenotype, reducing right ventricular systolic pressure (RVSP), vascular remodeling and right ventricular (RV) hypertrophy. Activation of {beta}3-AR with mirabegron improved pulmonary hemodynamics, reduced vascular remodeling and preserved RV function. {beta}3-AR activation promoted endothelial nitric oxide synthase (eNOS)-dependent NO production, indirectly inhibiting SMC proliferation. Additionally, {beta}3-AR activation improved mitochondrial fitness in endothelial cells by increasing uncoupling protein 2 (UCP2) expression, reducing reactive oxygen species (ROS) generation and preventing mitochondrial fragmentation. ConclusionsThese findings identify endothelial {beta}3-AR as a previously unrecognized regulator of pulmonary vascular homeostasis and provide a strong translational rationale for targeting the {beta}3-adrenergic pathway in PH. Given that mirabegron is already approved for clinical use, our results support its repurposing as a therapeutic strategy for pre-capillary forms of PH.

BackgroundCardiometabolic heart failure with preserved ejection fraction (HFpEF) is associated with systemic and cardiac inflammation and diastolic dysfunction. A-kinase anchoring protein 6{beta} (AKAP6{beta}) is a scaffold protein located at the cardiomyocyte outer nuclear membrane that promotes pathological cardiac remodeling via the recruitment of multiple regulatory proteins including protein kinases. In mice, adeno-associated virus (AAV) mediated expression of a peptide based upon a kinase binding domain (KBD) within AKAP6{beta} inhibited the development of heart failure due to chronic pressure overload. Whether KBD expression can also inhibit the development of cardiometabolic heart failure is unknown, and if so, the mechanism of KBD action in HFpEF has yet to be explored. MethodsThe efficacy of a cardiotropic self-complementary AAV gene therapy that expresses the AKAP6{beta} KBD peptide (AAV9sc.KBD) was tested in a female Ossabaw swine model of cardiometabolic syndrome and HFpEF. Single nucleus and bulk RNA sequencing of swine heart tissue and immunoprecipitation-mass spectrometry, live cell imaging, and biochemical assays using primary rat cardiomyocytes were employed to study KBD mechanism of action. ResultsAAV9sc.KBD inhibited the development of diastolic dysfunction and heart failure in the Ossabaw model, without negatively impacting systolic function. The improvement in cardiac phenotype was associated with decreased T-cell myocardial infiltrates and partial reversal of pathological gene expression. An unbiased interactome study revealed that the KBD peptide binds Ca2+/calmodulin-dependent protein kinase II (CaMKII), identifying CaMKII as a new AKAP6{beta} binding partner. Perinuclear CaMKII activity detected by live cell imaging required AKAP6{beta} expression and was inhibited by KBD expression. In addition, the CaMKII substrate Inhibitor of NF-{kappa}B Kinase {beta} (IKK{beta}) bound AKAP6{beta}. IKK phosphorylation in the Ossabaw model and in myocytes was inhibited by KBD expression, and NF-{kappa}B nuclear translocation in myocytes was dependent upon AKAP6{beta}-CaMKII protein complex formation. AAV9sc.KBD treatment inhibited cardiomyocyte NF-{kappa}B-dependent gene expression in the Ossabaw model. ConclusionsRegulated by perinuclear AKAP6{beta}-CaMKII signalosomes, NF-{kappa}B pro-inflammatory gene expression in cardiomyocytes participates in a positive feedback loop with cardiac inflammation promoting HFpEF. Proof-of-concept is provided in a large animal model that gene therapy-based cardiomyocyte expression of the KBD peptide will prevent cardiac dysfunction in cardiometabolic syndrome. Clinical PerspectiveO_ST_ABSWhat is newC_ST_ABSO_LIThe cardiomyocyte-selective gene therapy AAV9sc.KBD, which targets signalosomes organized by the scaffold protein AKAP6{beta}, is shown to inhibit myocardial T-cell infiltration and improve cardiac structure and function in a large animal model of cardiometabolic HFpEF. C_LIO_LIThe AKAP6{beta} KBD peptide is shown to bind and inhibit the function of CaMKII. C_LIO_LICaMKII and IKK{beta} are shown to participate in perinuclear AKAP6{beta} signalosomes, where they regulate activation of the NF-{kappa}B pro-inflammatory gene regulatory pathway. C_LI Clinical implicationsO_LIProof-of-concept for a novel strategy for the treatment of HFpEF is provided, intracellular expression by a cardiomyocyte-selective gene therapy vector of an inhibitory peptide, which will inhibit compartmentalized intracellular signal transduction. C_LIO_LIIn conjunction with previous studies in small rodents, the new data obtained in Ossabaw swine support clinical translation of the AAV9sc.KBD gene therapy. C_LI

Background Perioperative incidents such as hypoxic cardiac injury often have subtle or nonspecific clinical manifestations. Reduction in myocardial oxygenation precedes biochemical changes, as well as electrical and functional changes. Photoacoustic imaging (PAI) is a modality that uses laser irradiation of tissue to generate ultrasonic waves, enabling spatially resolved quantitative mapping of oxygenated and deoxygenated haemoglobin. We investigated the utility of PAI for real-time monitoring of myocardial and great vessel oxygenation. Methods Male CD-1 mice were anaesthetised, and photoacoustic and simultaneous B-mode images were acquired of the myocardium and right ventricular outflow tract (RVOT), the pulmonary artery, and aorta. PAI was performed at fractional inspired oxygen levels (FiO2) of 100%, 21%, and then 10%. Separate cohorts of mice were exposed to increasing intravenous doses of either combined phenylephrine and isoprenaline, or individual administration of vasoactive or adrenergic agents. Results PAI reliably distinguished changes in oxygenation in the RVOT cavity, pulmonary artery, aorta, and myocardium. PAI detected hypoxia-induced changes in oxygenation, revealing greater desaturation in the myocardium than in the RVOT (-9.85%, 95% CI -14.94 to -4.77, P<0.0001). Escalating doses of phenylephrine and isoprenaline caused a progressive desaturation of the myocardium and RVOT (mean [95% CI]; myocardium 16 mg/kg: -14.64% [-27.62 to -1.65], P=0.0038 and RVOT 32 mg/kg: -18.71% [-32.15 to -5.27], P=0.0003). Myocardial deoxygenation was detected before changes in systolic function or electrical abnormalities. Conclusions This work demonstrates that PAI can reliably monitor cardiac oxygen desaturation, potentially offering an earlier warning of cardiac dysfunction and injury compared to existing monitoring tools. Keywords: Echocardiography, hypoxaemia, hypoxia, myocardial injury, oxygenation, perioperative monitoring, photoacoustic imaging

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.

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.

A central unresolved and highly contested question in cardiac biology is whether the adult mammalian heart, believed to have a very limited endogenous cardiomyocyte (CM) regenerative capacity, can be coaxed into an effective regenerative response after acute CM loss. Using TgMyh6MCM:R26stop-DTA mice, we show that selective diffuse ablation of [~]15% of left ventricular CMs causes acute heart failure but is followed by complete structural and functional recovery within 28 days. Recovery is accomplished by robust generation of new mononucleated CMs, replacing [~]1/10 of the left ventricular CM compartment. This CM regeneration is produced by the activation of resident cardiac stem cells (CSCs), which exit quiescence, proliferate, produce new CMs, and subsequently return to quiescence. Depletion of the putative CSCs blocks repair, whereas transplantation of either clonogenic or primary CSCs through the systemic circulation fully restores myocardial regeneration and function, establishing that the CSCs home, nest and differentiate in the damaged myocardium and, therefore, are the main effectors of regeneration in this setting. Mechanistically, we show that miR-221-dependent repression of p57 governs the transition from quiescence--to activation--to differentiation--to quiescence of the CSCs, defining a reversible regulatory program which, under the proper conditions, endows the adult myocardium with robust CM regenerative competence.