Cardiovascular Research

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.

BackgroundSuccinate has emerged as an immunometabolic mediator of cardiovascular diseases. However, the enzymatic mechanisms linking macrophage succinate metabolism to aortic dissection remain incompletely understood. This study investigated whether Suclg2, which encodes the GDP-forming {beta}-subunit of succinyl-CoA ligase, regulates succinate-associated macrophage remodelling and aortic dissection progression. MethodsSuclg2 expression was examined in BAPN-induced AD and human acute type A aortic dissection tissues by Western Blot and immunofluorescence. Myeloid- and smooth muscle cell-specific Suclg2 conditional knockout mice were subjected to BAPN treatment to evaluate survival, aortic outcomes, histological injury and aortic morphology. Aortic RNA-seq was used to discover transcriptional changes. Bone marrow-derived macrophages were analysed under basal, M1-like and M2-like conditions to assess macrophage-intrinsic transcriptional responses. Plasma succinate levels and untargeted metabolomic profiles were further examined. ResultsSuclg2 was increased in murine and human dissected aortas and partially localized to CD68 cells. Myeloid Suclg2 deletion markedly reduced BAPN-induced aortic rupture and dissection, whereas smooth muscle cell Suclg2 deletion did not confer comparable protection. Aortic transcriptomic analysis showed that myeloid Suclg2 deficiency attenuated inflammatory adhesion and matrix-destructive programmes. In macrophages, Suclg2 deletion did not induce a simple M1/M2 polarization shift; instead, it remodelled lipid-handling, phagolysosomal, adhesive and matrix-remodelling pathways across stimulation states. Metabolic profiling showed reduced circulating succinate and broader changes in central carbon, lipid-associated, nucleotide and redox-related metabolites after myeloid Suclg2 deletion. ConclusionsMyeloid Suclg2 is a succinate-associated immunometabolic regulator of aortic dissection. Its deficiency protects against aortic dissection by reshaping macrophage inflammatory-remodelling programmes and the systemic metabolic environment.

BackgroundAtherosclerosis is a chronic inflammatory vascular disorder with persistent residual inflammation even after standard lipid-lowering therapy. Mounting evidence from bench to bedside suggests that diabetes and obesity accelerate atherosclerosis development. Tirzepatide (TZP), a dual Glucagon-Like Peptide-1 Receptor/Glucose-Dependent Insulinotropic Polypeptide Receptor (GLP-1R/GIPR) agonist approved for treating diabetes and obesity, has demonstrated proven cardiometabolic efficacy in large cardiovascular outcome trials. However, it remains largely uncertain whether TZP attenuates atherosclerosis independent of its anti-diabetic and anti-obese effects through direct actions on the vasculature. MethodsWe established atherosclerotic mouse models under diabetic, obese, and non-diabetic/non-obese conditions. Analysis of covariance (ANCOVA) and pair-feeding experiments were applied to experimentally decouple weight-dependent metabolic improvement from intrinsic vasculoprotection. Molecular and cell biological assays in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were performed to dissect the underlying signaling mechanisms. ResultsTZP markedly reduced aortic plaque burden and inflammation, restrained necrotic core enlargement, and improved plaque stability across all experimental mouse models. Both ANCOVA and pair-feeding experiments confirmed that these atheroprotective effects were independent of food intake and body weight loss. Furthermore, TZP attenuated systemic and vascular inflammation in Tumor Necrosis Factor- (TNF)-treated C57BL/6J mice, and this protection occurred without changes in body weight or blood glucose levels. Mechanistically, TZP directly targeted endothelial cells and activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/endothelial nitric oxide synthase (eNOS) pathway, increased eNOS phosphorylation and nitric oxide bioavailability, consequently downregulating the expression of the pro-inflammatory adhesion molecules Vascular Cell Adhesion Molecule-1 (VCAM-1) and Intercellular Adhesion Molecule-1 (ICAM-1). ConclusionsTZP arrests atherosclerosis progression through weight loss-independent anti-inflammatory mechanisms. These findings implicate TZP as a promising therapeutic drug for mitigating residual vascular inflammation in patients with atherosclerotic cardiovascular disease (ASCVD), irrespective of glycemic status or obesity. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LITirzepatide exerts direct anti-atherosclerotic effects in preclinical mouse models of atherosclerosis under diabetic, obese, and non-obese conditions. C_LIO_LITirzepatide directly targets endothelial GLP-1R/GIPR and downstream cAMP/PKA/eNOS signaling pathway to suppress NF-{kappa}B-driven vascular inflammation, thereby uncovering a previously unrecognized vasculoprotective mechanism underlying its cardiovascular benefits C_LI What Are the Clinical Implications?O_LITirzepatide exerts direct vascular protective effects independent of body weight reduction, suggesting that its cardiovascular benefits may extend beyond glycemic control and obesity management. C_LIO_LITirzepatide may represent a promising therapeutic drug for addressing residual vascular inflammation in ASCVD patients, including those without overt diabetes or obesity C_LI

ObjectiveChronic activation of the renin-angiotensin-aldosterone system (RAAS) promotes pathological remodeling of both myocardium and coronary arteries, yet the mechanisms that distinguish myocardial from vascular remodeling remain poorly defined. This study dissects the relative contributions of hemodynamic versus neurohumoral stress to cardiac remodeling, with emphasis on coronary vasculopathy and vascular smooth muscle cell (VSMC) plasticity. MethodsThree murine models were used: transverse aortic constriction (TAC), angiotensin II (AngII) plus phenylephrine (AngII/PE), and high-dose angiotensin II (HD-AngII). Hemodynamics were assessed by catheterization at early and late time points. Histological and immunostaining analyses quantified interstitial and perivascular remodeling, including cardiomyocyte hypertrophy, interstitial and perivascular fibrosis, VSMC phenotype transitions, proliferation and quiescence markers, and neointimal and elastic lamina remodeling. ResultsAfter 28 days, all models exhibited diastolic dysfunction and myocardial fibrosis. Systolic pressure averaged [~]130 mmHg in both AngII models versus [~]200 mmHg in TAC. Despite lower pressure, myocardial fibrosis was greater in AngII/PE and HD-AngII models. While TAC induced uniform cardiomyocyte hypertrophy, hypertrophy in AngII models localized near fibrotic and perivascular regions. Increasing AngII dosage shifted remodeling from predominantly myocardial to predominantly vascular phenotypes, accompanied by VSMC dedifferentiation, proliferation, centripetal migration across the internal elastic lamina, neointima formation, elastic lamina disruption, and increased circulating desmosine, consistent with elastin degradation. AKT signaling was selectively increased in coronary VSMCs during this vasculopathic remodeling. Lineage-tracing analyses showed that Ang II-driven coronary neointima formation occurs beneath an intact endothelial monolayer and is composed predominantly of VSMC-derived cells, highlighting a VSMC-centric vasculopathy distinct from classic endothelium-initiated vascular remodeling. ConclusionHemodynamic pressure overload and AngII-dominant neurohumoral stress drive distinct cardiac remodeling phenotypes: TAC primarily elicits uniform myocardial hypertrophy and interstitial fibrosis, whereas chronic AngII exposure preferentially promotes a VSMC-centric coronary vasculopathy with perivascular fibrosis and elastic lamina injury at lower pressure load. These complementary models help distinguish pressure-dependent from AngII-mediated vascular mechanisms and provide a platform to develop targeted therapies for coronary vasculopathy and AngII-driven vascular disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/733633v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@469202org.highwire.dtl.DTLVardef@11bde2borg.highwire.dtl.DTLVardef@96ea08org.highwire.dtl.DTLVardef@1deca16_HPS_FORMAT_FIGEXP M_FIG C_FIG

Bradycardia occurs when the heart rate is lower than normal resulting in reduced cerebral blood flow and contributing to neurodegeneration in adults but how it affects embryonic cerebrovascular development is not well studied. We induce bradycardia by targeting the heart pacemaker channel Hcn4 via chemical (ivabradine) and genetic (hcn4 mutant) methods. Bradycardia results in reduced brain vessel diameter and mural cell (pericyte and vascular smooth muscle cell) number. Endothelial cells are the first responders in sensing changes in blood flow, and we show that signalling through the canonical endothelial-autonomous mechanosensitive pathway (Piezo1, Mek5, Erk5, Klf2) is reduced in bradycardia. To identify the ligand-receptor combination that transmits signals to developing mural cells, we show that expression of the Notch ligand jagged2b is decreased in the brain of both hcn4 and klf2 mutants. jag2b knockdown reduces mural cell numbers in brain vessels. Restoring jag2b levels increases mural cell numbers in both wildtype and hcn4 mutants. Our work connects bradycardia, mechanosensitive signaling and mural cell recruitment demonstrating that mural cell numbers can be increased in bradycardia by restoring Notch signalling via upregulating endothelial Jag2b. SummaryBradycardia models show reduced blood flow, Piezo1-klf2-jag2b-notch3 mechanosensing and mural cell recruitment to developing brain vasculature. Restoration of jag2, an endogenous endothelial cell ligand, rescues mural cell numbers in bradycardia mutants.

Background: Abdominal aortic aneurysms (AAA) are characterized by dilation of the aorta that can lead to aortic rupture and death. The transcriptional co-activators Yes-Associated Protein (YAP) and WW-domain-containing transcriptional co-activator with PDZ-binding motif (TAZ) are mechanosensitive effectors of the highly conserved Hippo signaling pathway. It is hypothesized that cell-specific YAP/TAZ signaling in endothelial cells (EC) plays a pivotal role in mediating AAA formation and rupture. Methods: Single-cell RNA-sequencing in human AAAs was performed and differentially expressed genes (DEGs) were identified in the endothelial cell cluster. YAP/TAZ mRNA and protein expression were also assessed in human AAA and control aortic tissue. Two established murine AAA models were used with male C57BL/6 and EC-CreERT2-YAPfl/fl/TAZfl/fl mice with/without Verteporfin (VPF, YAP/TAZ inhibitor) and XMU-MP-1 (YAP/TAZ activator) treatments. On postoperative days 14 and 28, aortic diameter, histology, cytokine, and MMP2 expressions were evaluated. Results: A significant alteration in EC-specific differentially expressed YAP/TAZ-related genes was observed in which 242 genes were upregulated and 71 genes were downregulated in AAA compared to controls. Human AAA tissue showed a significant increase in YAP and TAZ protein expressions compared to controls. Elastase-treated EC-YAP/TAZ-/- mice showed a significant decrease in AAA diameter compared to littermate controls. Histological quantification revealed preservation of -smooth muscle actin, reduced elastin fiber breaks, and decreased macrophage infiltration in EC-YAP/TAZ-/- mice compared to littermate controls. Importantly, pharmacological inhibition of YAP/TAZ using VPF significantly attenuated AAAs in two experimental murine models. In vitro data demonstrates that VPF inhibits endothelial cell YAP expression, downregulating pathways associated with pathogenic angiogenesis and vascular inflammation. Conclusions: These data suggest that EC-specific YAP/TAZ signaling mediates AAA formation. Pharmacological inhibition of the Hippo pathway can significantly mitigate aortic inflammation and vascular remodeling to decrease the progression of AAAs and prevent aortic rupture.

Background: Non-monogenic arteriopathies are often classified as distinct entities according to the arterial territory involved, yet they share clinical features and may co-occur in the same individual. This pattern suggests shared susceptibility across anatomically distinct arteriopathies, potentially driven by common biological and genetic mechanisms. Methods: We investigated the shared genetic architecture of five arteriopathies (cervical artery dissection (CeAD), intracranial aneurysm (IA), spontaneous coronary artery dissection (SCAD), aortic aneurysm and dissection (AAD), and fibromuscular dysplasia (FMD)) using LD score regression, Association analysis based on SubSETs (ASSET), pairwise Multi-Trait Analysis of Genome-wide association summary statistics (MTAG), pleiotropy mapping and Mendelian randomization (MR) to identify shared loci and prioritise candidate causal genes. Results: LD score regression identified significant positive genetic correlations between CeAD-SCAD (rg = 0.64), IA-AAD (rg = 0.33), IA-SCAD (rg = 0.37), CeAD-AAD (rg = 0.56) and SCAD-AAD (rg = 0.20). ASSET identified 37 shared independent loci, and in MTAG analyses, one novel locus was identified for CeAD and SCAD (SLC39A8) and one for IA (FGF5). 13 loci showed strong cross-trait colocalization, including PHACTR1, LRP1, and CDKN2B-AS1. Using the Genotype-Phenotype Map, we found that arteriopathy-associated variants colocalized with blood pressure- and migraine-related traits, while many showed effect directions opposite to those observed for coronary artery disease. Proteome-wide MR identified 67 circulating proteins associated with at least one trait, including ECM1 and SHISA5 for CeAD and FGF5 for IA, with 17 supported by colocalization. Transcriptome-wide MR identified 204 colocalized tissue?specific signals, of which, 14 were shared across multiple traits. Enrichment analyses implicated pathways related to vascular development, smooth muscle cell function, extracellular matrix organization, and TGF-? signaling. Conclusions: These findings support shared genetic architecture across anatomically distinct arteriopathies, implicating pathways involved in vascular structure and prioritising therapeutic targets for future mechanistic investigation.

The insulin-like growth factor (IGF)/IGF-binding protein (IGFBP) axis has been implicated in diabetes mellitus and the associated burden of cardiovascular complications. Higher circulating levels of IGFBP-1 and IGFBP-2 have been established as markers of protection from incident type 2 diabetes, yet their associations with cardiovascular disease remain unclear. Utilising the UK Biobank (UKB) resource to integrate disease outcomes, plasma proteomics and MRI data, we examined associations of IGFBP-1 and IGFBP-2 with incident diabetes and cardiovascular disease. Approximately 50,000 UKB participants with plasma proteomic measurements for IGFBP-1 and IGFBP-2 were included. Multivariate Cox regression models revealed that participants in the highest quartiles of IGFBP-1 and IGFBP-2 had a substantially lower risk of incident diabetes (hazard ratio (HR) = 0.31 and 0.32 respectively), but, paradoxically, had increased risks of incident macrovascular disease, all-cause and cardiovascular-related mortality (HR = 1.81 and 2.39). Both proteins were negatively associated with HbA1c levels, triglyceride/HDL ratio and abdominal adiposity, yet positively associated with NT-proBNP, troponin I, cardiac chamber size and aortic dimensions. In summary, negative associations of IGFBP-1 and IGFBP-2 with incident diabetes mellitus did not translate to a reduced cardiovascular risk, suggesting potentially complex actions of IGFBP-1 and IGFBP-2 in the pathophysiology of cardiometabolic disease.

Background Carotid plaque calcification is commonly interpreted as a marker of atherosclerotic burden, but its prognostic meaning may depend on calcification morphology. Whether histologically defined calcification subtypes in carotid plaques identify systemic cardiovascular disease and long-term cardiac risk remains unclear. Methods We studied 479 patients, including 154 women, undergoing carotid endarterectomy in the Helsinki Carotid Endarterectomy Study 2 with 10-year follow-up. Hematoxylin-eosin-stained plaque sections were digitized, and artificial intelligence-based image analysis was used to quantify sheet, nodular, and total calcification as proportions of plaque tissue area. The primary endpoint was cardiac death; secondary endpoints were major coronary events and major adverse limb events (MALE). Associations between calcification tertiles and outcomes were assessed using Fine-Gray regression models adjusted for conventional cardiovascular risk factors, with prespecified sex-stratified analyses. Results Total calcification was associated with baseline coronary artery disease and chronic heart failure but did not predict cardiac mortality. In contrast, nodular calcification was independently associated with cardiac death. Compared with the lowest tertile, the middle and highest tertiles of nodular calcification were associated with increased cardiac mortality in the whole cohort (subdistribution hazard ratio [sHR], 2.87 [95% CI, 1.39-5.91] and 2.55 [95% CI, 1.24-5.27], respectively). Nodular calcification was also associated with baseline peripheral artery disease. High sheet calcification was associated with cardiac death in men (sHR, 2.3 [95% CI, 1.0-5.1]) but not in women. Exploratory sex-stratified analyses suggested that high total calcification was associated with major coronary events and MALE in women, but not in men. Conclusions Carotid plaque calcification morphology, rather than total calcification burden alone, is associated with long-term cardiac mortality after carotid endarterectomy. Nodular calcification emerged as the strongest prognostic phenotype, while exploratory sex-stratified findings suggest that total calcification may reflect coronary and peripheral event risk in women.

Rupture of the fibrous cap is the primary cause of clinical complications from atherosclerosis. Smooth muscle cells (SMCs) are a major contributor to fibrous cap development and stability through de-differentiation to extracellular matrix-producing cells. We previously showed that the antioxidant enzyme cytoglobin (CYGB) is expressed in vascular SMCs and regulates SMC dependent vascular remodeling and gene expression. In the present study, we investigated the function of SMC-CYGB in atherosclerosis. To this end, we generated a mouse line with SMC-specific deletion of Cygb and simultaneous SMC-lineage tracing. We found that SMC specific deletion of CYGB increased fibrous cap thickness in a 17-week PCSK9-AAV8 gain of function combined with Western diet mouse model of atherosclerosis. SMC specific deletion of CYGB increased collagen deposition and SMC cellularity of the fibrous cap in the absence of changes in total plaque and necrotic core sizes. CYGB expression in SMCs was associated with transdifferentiation towards a fibroblast-like, matrix remodeling phenotype. Finally, CYGB was expressed in the fibrous cap of human coronary atherosclerotic lesions and was associated with ACTA2 positive cells. These results provide first-time evidence that SMC-CYGB reduces plaque stability by decreasing cap thickness, collagen deposition, and SMC cellularity.

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.

Abdominal aortic aneurysms (AAAs) occur predominantly in the elderly population and currently there is no effective pharmacological therapy for mitigating AAA growth and preventing impending rupture. Proprotein subtilisin kexin type 9 (PCSK9) gene has been identified as a specific risk-locus for AAA development. However, the mechanistic and clinical role of PCSK9-mediated signaling in AAAs has not been delineated. We demonstrate that treatment with PCSK9 inhibitors, such as Evolocumab, mitigates vascular inflammation and remodeling, resulting in attenuated aneurysm growth in clinical datasets as well as experimental models of AAA and aortic rupture. Mechanistically, Evolocumab immunomodulates macrophage reprogramming to enhance clearance of apoptotic smooth muscle cells via MerTK-dependent efferocytosis that ameliorates aortic inflammation and vascular remodeling. Furthermore, Evolocumab increases the expression of oxidized phosphatidylserine species and decreases expression of lysophospholipids, succinate, and glycolytic intermediates within the aortic wall compared to untreated controls, further enhancing the pro-resolving functions of macrophages. Collectively, our data demonstrates the ability of PCSK9 inhibition to regulate macrophage-specific efferocytosis that limits AAA progression and prevents aortic rupture.

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.

AimsWhile the muscle-enriched microRNA-378a (miR-378a) has been implicated in cardiac hypertrophy and stress responses, its role in maintaining cardiomyocyte metabolic homeostasis, mitochondrial function, and angiogenic paracrine signaling under physiological and post-injury conditions remains unclear. This study addresses these gaps by examining the molecular and functional consequences of miR-378a deficiency in murine heart and human cardiomyocytes. Methods and ResultsCardiac structure and function were analyzed in miR-378a-deficient (miR-378a-/-) and wild-type (miR-378a+/+) mice at 12 weeks and 17 months of age, revealing that miR-378a loss promoted myocardial fibrosis, altered IGF1R-AKT signaling, and impaired cardiac performance, with age-dependent effects. Integrated transcriptomic and proteomic analyses in miR-378a-/- and control mice, as well as in human iPSC-derived cardiomyocytes (hiPSC-CM) of both genotypes, revealed deregulated pathways related to translation, metabolism, and cardiomyopathy-associated signaling. In hiPSC-CM, miR-378a knockout (KO) impaired mitochondrial respiration, disrupted mitochondrial morphology, and reduced mitochondrial DNA content, accompanied by altered mitophagy and biogenesis. KO cells also showed increased glucose uptake but reduced glycogen storage, accompanied by changes in key metabolic regulators, and displayed diminished angiogenic potential. Finally, hiPSC-CM overexpressing miR-378a were delivered in a mouse model of acute myocardial infarction, but overexpression did not further enhance their therapeutic effect. ConclusionsThis study broadens our understanding of miR-378as physiological role in murine hearts and human cardiomyocytes, demonstrating its impact on contractility, mitochondrial integrity, glucose metabolism, and angiogenic paracrine signaling. However, overexpression of miR-378a in hiPSC-CM offers limited additional benefit in cell therapy for acute myocardial infarction.

Our prior work demonstrated that lowering dietary branched-chain amino acids (BCAAs) improves cardiac outcomes during pressure overload-induced stress. Here, we identify isoleucine restriction (IleR) as the key driver of this effect. Dietary isoleucine restriction induces hypophagia and weight loss, recapitulating the effects of caloric restriction (CR). Although it does not prevent the initial development of left ventricular hypertrophy, it halts its progression and the decline in ejection fraction compared with controls. This is associated with preservation of electron transport chain (ETC) gene expression, cristae structure, NAD+/NADH levels, and mitochondrial respiratory capacity in cardiomyocytes, which is recapitulated by CR. Mechanistically, both IleR and CR diets increase Foxo3 expression, thereby blocking the decline in expression of its target ETC and mitochondrial genome-encoded genes. Consequently, this improves mitochondrial respiratory capacity and reduces cardiac fibrosis. We conclude that restricting dietary isoleucine improves cardiac health by increasing Foxo3 expression and mitochondrial function via a cell-autonomous mechanism and by reducing caloric intake.

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

Background and Aims Current diagnostic criteria for metabolic syndrome (MetS) may inadequately capture underlying metabolic heterogeneity and associated cardiovascular risks. We aimed to use expanded cardiometabolic variables to identify new cardiometabolic phenogroups with relevance to prognosis and risk stratification. Methods Latent class analysis (LCA) was applied to a discovery cohort (RESET; n=1,034), using the six conventional MetS measures and eight additional variables. A decision tree model was constructed using the most important variables to enable practical phenogroup classification and facilitate external validation. External validation was conducted in three independent cohorts, PICMAN (n = 120), UK Biobank (n = 344,817), and CHARLS (n = 12,145), analysing for proteomic signatures and cardiovascular outcomes. Results Five latent phenogroups were identified in the discovery cohort: Metabolically Preserved with and without isolated hypertension (each n=244; 23.6%), Lean-Insulin Resistant (IR) (n=140; 13.5%), Obese-Insulin Sensitive (IS) (n=211; 20.4%), and Obese-IR (n=195; 18.9%). Lean-IR and Obese-IS showed discordant adiposity and insulin/glycemic status, and a low prevalence of MetS (21.4% and 31.3%, respectively), whereas MetS was high (75.9%) only in the Obese-IR group. A decision tree model using four binary indicators (visceral adiposity, IR, elevated SBP, and HbA1c) accurately classified individuals into the five latent phenogroups and was subsequently deployed for external validation. Validation in PICMAN showed significantly higher liver fat (Mean 9.0% [SD 6.3%]) in Lean-IR versus Metabolically Preserved (Mean 2.8% [SD 1.8%], P=0.002). Plasma proteomic analyses further reflected unique metabolic-inflammation signatures across the 5 groups. Validation in the UK Biobank showed significant association between the latent phenogroups with outcomes of myocardial infarction and stroke. Hazard ratios for the composite outcome after adjusting for age and sex were 1.52 (95% CI, 1.43-1.61) for isolated hypertension, 1.86 (1.75-1.98) for Lean-IR, 1.85 (1.75-1.97) for Obese-IS, and 2.75 (2.56-2.95) for Obese-IR, compared with the Metabolically Preserved group. Conclusion Expanded cardiometabolic risk factors reveal metabolic heterogeneity obscured by current MetS criteria. Incorporating visceral adiposity and IR into a novel classification system refines cardiovascular risk stratification for the management of cardiometabolic disease.

The cellular origin of smooth muscle cell (SMC)-derived populations in vascular lesions remains unresolved. Here we show, using single-cell transcriptomic analyses spanning carotid ligation injury, Myh11-CreERT{superscript 2}-traced aortic homeostasis, and LDLR- and ApoE-deficient atherosclerosis, that a rare progenitor-like "Primed" SMC compartment pre-exists at baseline in all models and in the healthy human aorta. Relative to contractile SMCs, Primed SMCs attenuate sarcomeric and contractile programmes while inducing matricellular, progenitor-niche and chondrogenic-poised developmental programmes, resolving into conserved niche/progenitor (Cd34, Fst, Tnfrsf11b) and matricellular (Vcam1, Thbs1, Timp1) cores overlaid by vessel-specific signatures, on a retained SMC identity. Multiple orthogonal computational lineage-inference approaches indicate that this compartment expands predominantly through autonomous self-renewal and is the dominant inferred source of cycling and lesion fibrochondrocyte populations, while contractile SMCs are consistently depleted as a feeder source. These findings reframe lesional SMC cellularity as expansion of a pre-existing Primed compartment rather than widespread phenotypic switching of contractile SMCs.

Tricuspid valve regurgitation is a frequent valve lesion and, if severe, an independent predictor of mortality. In most patients, the valve itself has historically been considered intact. Yet, we have previously shown that the valve may not be an innocent bystander. In multiple sheep models, we have shown that the tricuspid valve thickens and stiffens. This remodeling may contribute to valve disease. Our goal is to extend our investigation of tricuspid valve remodeling to a rodent model, potentially opening scientific opportunity and enabling scaling our studies. To this end, we used pulmonary artery banding (PAB) in male rats to induce pressure overload and right ventricular remodeling. After excising the tricuspid valve, we quantified anterior leaflet morphology, mapped anterior leaflet thickness using optical coherence tomography, and evaluated anterior leaflet belly mechanics using a custom bulge testing system. Compared with SHAM controls, PAB increased anterior leaflet area. Moreover, anterior leaflets in PAB animals exhibited region-specific thickening, with the largest increases near the annulus. Finally, anterior leaflets in PAB animals were significantly less compliant. However, leaflet stiffening stemmed from aforementioned thickening, i.e., structural stiffening, not constitutive stiffening. Our findings demonstrate that we can reliably quantify leaflet area, thickness, and stiffness in the minuscule tricuspid valves of rats. We also show that tricuspid valve remodeling is not ovine-specific, but also affects the tricuspid valves of rats. Together, our findings support our hypothesis that tricuspid valves are not innocent bystanders in regurgitation, and that rats may serve as a scalable model system for future investigations. NEW & NOTEWORTHYUsing a rat pulmonary artery banding model of pulmonary hypertension, we show that chronic right ventricular pressure overload induces leaflet enlargement and region-specific thickness remodeling of the tricuspid valve. Although structural mechanical metrics change under pressure loading, normalization by thickness reveals that geometric remodeling rather than intrinsic material stiffening predominates. These findings highlight leaflet structural (mal)adaptation as a potential contributor to functional tricuspid regurgitation and underscore the importance of considering leaflet geometry in therapeutic strategies.