JACC: Basic to Translational Science

BackgroundMarfan syndrome (MFS) is a life-threatening heritable connective tissue disorder caused by pathogenic variants in fibrillin-1, characterized by progressive cardiovascular disease. Current medical therapies slow disease progression but do not prevent major complications, underscoring the need for new treatment strategies and unbiased discovery approaches. MethodsWe used a zebrafish model of MFS lacking fibrillin-3 (fbn3-/-), which recapitulates key cardiovascular phenotypes including cardiac stress, valvular defects, arrhythmia, and aortic dilation. To enable sensitive, quantitative assessment of cardiac stress, we generated a novel transgenic zebrafish reporter expressing secreted nanoluciferase under control of the stress-responsive nppb promoter. This reporter was combined with morphological phenotyping and bulbus arteriosus (BA) imaging. We evaluated standard MFS therapies, targeted modulators of TGF-{beta} signaling, and performed an unbiased high-throughput drug screen of over 1 500 clinically approved compounds across multiple developmental treatment windows. Resultsfbn3-/- larvae exhibited markedly elevated nppb activity that correlated with phenotypic severity and peaked during stages of highest mortality. The nanoluciferase reporter provided a [~]1 000-fold dynamic range, substantially outperforming Firefly luciferase-based assays. Pharmacological inhibition of TGF-{beta} signaling produced transient or deleterious effects, while {beta}-blockers, losartan, and allopurinol failed to consistently improve cardiac stress, pericardial edema, or BA dilation. The unbiased high-throughput drug screen identified a small number of primary and secondary hits; however, none demonstrated reproducible phenotypic rescue upon rigorous multi-dose, multi-time window validation. ConclusionsThis study establishes a sensitive zebrafish-based platform for early, quantitative assessment of cardiovascular stress in MFS. Our findings highlight the limited efficacy of current therapies, the context-dependent nature of TGF-{beta} modulation, and the biological complexity underlying MFS pathogenesis. Although no definitive therapeutic candidates were identified, this work lays a robust foundation for expanded unbiased discovery efforts aimed at identifying disease-modifying interventions for MFS.

Bicuspid aortic valve (BAV) is one of the most common congenital heart defects but its genetic basis remains incompletely defined. Extracellular matrix components play key roles in outflow tract (OFT) and valve development, but their contribution to BAV is not fully established. Following the analysis of a cohort of BAV patients, we identified a family harbouring a rare human ELASTIN (ELN) variant (p.Gln691X). To assess its pathogenicity, we generated a zebrafish elna/b double knockout (KO) using an RNAless CRISPR Cas9 strategy to avoid genetic compensation. This mutant exhibited cardiovascular defects including OFT anomalies, reduced stroke volume and dysmorphic aortic valves, highlighting Elastins critical role in cardiac development. We then used this model to test the ELN variant identified in the BAV family. We found that wild-type ELN mRNA was able to restore normal cardiac function and morphology, whereas the variant ELN mRNA failed to do so. This study establishes a robust in vivo model to assess ELN variant pathogenicity and provides evidence linking ELASTIN to BAV, opening new avenues for uncovering the genetic mechanisms underlying BAV.

IntroductionHypertrophic Cardiomyopathy (HCM) is a disease defined by the development of left ventricle hypertrophy. One of the most commonly mutated genes in HCM is cardiac myosin binding protein C (MYBPC3). MYBPC3 protein localizes to the cardiomyocyte sarcomere, but studies have reported detection of both MYBPC3 RNA and protein in non-cardiomyocyte cell populations. Therefore, it was unclear if MYBPC3 expression in non-cardiomyocyte cell populations altered the development of cardiomyopathy caused by MYBPC3 protein deficiency. MethodsWe utilized genetically modified murine models with germline deletion of Mybpc3 exons 3 to 5 (Mybpc3-/-) or cardiomyocyte specific deletion of Mybpc3 exons 3 to 5 (Mybpc3fl/fl; Myh6-Cre). Gene expression was assessed using quantitative RT-PCR. Whole tissue protein levels were assessed using immunoblots. Immunohistochemistry and proximity ligation assays were performed to evaluate in situ protein expression. Echocardiography was utilized to measure left ventricular structure and function. ResultsMybpc3 mRNA was detected in multiple organs including the heart, lung and blood from both humans and mice. Utilizing transgenic murine models with germline or cardiomyocyte specific deletion of Mybpc3 exons 3-5, we discovered that the Mybpc3 mRNA detected in extracardiac locations originated primarily from cardiomyocytes. Likewise, MYBPC3 protein was identified in myocardial tissue but not in other organs and cardiomyocytes were the only cell population in myocardial tissue that had detectable MYBPC3 protein. Importantly, cardiomyocyte deletion of Mybpc3 caused similar pathological myocardial remodeling and alterations in left ventricular function compared to germline deletion of Mybpc3 in all cell populations. ConclusionsOur results show that cardiomyocytes are the primary cell source of Mybpc3 mRNA detected in extracardiac organs and they are the principal cell type responsible for the cardiomyopathy caused by MYBPC3 protein deficiency. These results suggest that selective targeting of cardiomyocytes should be the most efficient approach to treat cardiomyopathies associated with MYBPC3 deficiency.

AimsHypertrophic cardiomyopathy (HCM) is the most prevalent genetic heart disorder, characterized by significant phenotypic variability even among individuals with identical MYH7 mutations. This study aims to elucidate factors contributing to this variability and identify drivers of phenotype penetrance. We compared the baseline phenotypes of a highly penetrant MYH7 H251N mutation and the variably penetrant MYH7 G256E mutation and investigated the impact of adding beta-adrenergic stimulation and homozygosity on disease phenotype penetrance using cardiomyocytes from an isogenic line of human induced pluripotent stem cells (hiPSC-CMs). Methods and ResultsIsogenic hiPSCs with MYH7 H251N and MYH7 G256E mutations were generated using CRISPR/Cas9 technology and differentiated into cardiomyocytes (CMs). Single-cell RNA sequencing (scRNAseq) and functional analysis of contractile function revealed consistent HCM phenotype presentation in H251N CMs, whereas G256E CMs exhibited a subtle and more variable phenotype. Beta-adrenergic stimulation induced a distinct metabolic stress response in G256E CMs, characterized by impaired mitochondrial ATP upregulation. Increasing mutant gene dosage from hetero- to homozygosity led to consistent increase in hypertrophic and structural gene expression changes in G256E CMs at RNA and protein levels. These changes were distinct from the changes observed with stress response. Importantly, homozygous G256E CMs exhibited a hypercontractile functional and disorganized structural phenotype. Across multiple experimental conditions, we identified consistent increase in cardiomyocyte specific transcriptomic markers such as NPPB, APOE, PDLIM3 and ANKRD1. ConclusionsOur study highlights the use of a variably penetrant MYH7 mutation to investigate factors that influence HCM phenotype penetrance. Specifically, we found that mutant gene dosage and beta-adrenergic stimulation induce distinct HCM disease phenotypes, providing novel insights into mechanisms that may contribute to variable disease expression in HCM. Translational PerspectiveHCM is characterized by significant phenotypic variability, complicating both diagnosis and clinical management. This study explores the factors driving HCM phenotype penetrance using isogenic hiPSC-CMs with MYH7 mutations. We demonstrate that beta-adrenergic stimulation and increased mutant gene dosage significantly impact HCM disease penetrance. Beta-adrenergic stimulation triggers metabolic stress responses, while increased gene dosage leads to a hypercontractile and structurally disorganized phenotype. These findings provide insight into how specific modifiers can shape disease-associated phenotypes in HCM model systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/729411v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@7fbc4org.highwire.dtl.DTLVardef@220d2forg.highwire.dtl.DTLVardef@18fa366org.highwire.dtl.DTLVardef@13091d4_HPS_FORMAT_FIGEXP M_FIG C_FIG

The aorta, normally resilient to hemodynamic stresses, becomes vulnerable to structural failure due to diverse conditions that weaken the wall. We injected fluid into excised specimens of human ascending aorta with pressure monitoring to quantify the impact of clinical and histological factors on mural damage. Two modes of medial injury emerged with distinct pressure tracings. Extravasation was characterized by diffuse infiltration of fluid with widespread damage of smooth muscle cells and collagen fibers but limited separation of elastic lamellae. By contrast, delamination was characterized by marked separation of elastic lamellae along a single plane with damage to cells and fibrillar matrix restricted to adjacent laminae. Aging, aortic dilatation, and family history associated with lower pressures causing delamination, whereas a diagnosis of hypertension associated with higher pressures suggesting resilience to dissection. Collagen fraction adjacent to delamination correlated with higher pressures as did decreased smooth muscle cell density and increased glycosaminoglycan fraction, although several clinical and histological variables were interrelated. Protein cross-linking strengthened and enzymatic digestion of collagen weakened the aortic wall, while acute cell lysis with detergent had no effect. We conclude that increased functional medial collagen has an adaptive protective role in aortic remodeling rather than signifying medial degeneration.

I.BackgroundPulmonary arterial hypertension (PAH) is a debilitating cardiopulmonary disease characterized by progressive remodeling of the pulmonary vasculature. Pathologic transforming growth factor-{beta} (TGF-{beta}) signaling is an essential driver of vascular remodeling in PAH. While global inhibitors of TGF-{beta} exist, their clinical application is limited by systemic adverse effects. Therefore, a critically unmet need in PAH is to identify pulmonary vascular-specific regulators of the TGF-{beta} axis, which would selectively enhance clinical efficacy while minimizing adverse effects. As the clinical care of PAH largely promotes vasodilation, and only one FDA-approved agent targets vascular remodeling, this study aimed to identify selective, therapeutically targetable regulators of the TGF-{beta} axis in the PAH pulmonary vasculature. MethodsCD248 was identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics in human lungs. CD248 levels were assessed across human, rat, and mouse lung tissues using western blotting, RTqPCR, and/or immunofluorescence techniques. CD248-null (CD248-/-) mice were used to study the contribution of CD248 to hypoxia-sugen (H/S)-induced PAH. The mechanistic role of CD248 in PAH vascular remodeling and TGF-{beta} signaling was assessed by genetic (siRNA knockdown; overexpression) and pharmacologic (Ontuxizumab) manipulation of primary human pulmonary vascular cells. ResultsLC-MS/MS proteomics coupled with pathway enrichment analysis of human lung tissue identified CD248 as a putative mediator of vascular remodeling that is elevated in PAH lungs. CD248 was elevated in PAH pulmonary artery smooth muscle cells (PASMCs) across human, rat, and mouse lung tissue. CD248-/- mice were protected from H/S-induced elevations in right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary artery muscularization. CD248 knock-down reduced cell proliferation and migration of primary PAH PASMCs. CD248 was essential for phospho-activation of TGF-{beta} receptor I (T{beta}RI) at S165 and canonical phosphorylation of SMAD3 at S423/425. CD248 loss blunted TGF-{beta}-induced gene expression (FN1, Col11, -SMA) and activated expression of the vasoprotective matrix metalloprotease, MMP-8. Mechanistically, CD248 interacted with and enhanced de novo phosphorylation and stability of T{beta}RI, blocking its ubiquitin-mediated proteasomal degradation. Ontuxizumab promoted T{beta}RI instability and attenuated the production of FN1, Col11, and -SMA in primary PAH PASMCs. ConclusionsThis work identifies CD248 as a previously unrecognized co-activator of T{beta}RI in PAH. As CD248 is largely quiescent in most adult tissues yet pathologically upregulated in the PAH pulmonary vasculature, this study supports the potential of anti-CD248 therapy as a novel pulmonary vascular-specific alternative to systemic TGF-{beta} inhibition.

Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous condition with incompletely defined myocardial mechanisms. Here, using a two-hit murine model of cardiometabolic HFpEF induced by high-fat diet and endothelial nitric oxide synthase inhibition, we define a mitochondrial metabolic phenotype characterized by substrate inflexibility, redox stress, and S-nitrosylation remodeling. While global proteomic changes were modest, metabolomic profiling revealed accumulation of tricarboxylic acid cycle intermediates, increased dicarboxylic acids, and altered redox-associated metabolites, consistent with inefficient oxidative metabolism and mitochondrial redox imbalance in this experimental setting. S-nitrosylation proteomics demonstrated a highly organized and bidirectional remodeling pattern affecting proteins involved in fatty acid/lipid metabolism, carbohydrate metabolism, mitochondrial energy metabolism, amino acid and organic acid metabolism, nucleotide/cofactor metabolism, and redox defense. Beta-hydroxybutyrate (BHB), an alternative mitochondrial substrate, improved basal and ATP-linked respiration, reduced selected TCA-cycle intermediates, lowered mitochondrial reactive oxygen species and the NADH/NAD+ ratio, partially restored the GSH/GSSG ratio, and improved diastolic and functional phenotypes without altering preserved ejection fraction. Together, these findings define a redox-sensitive mitochondrial metabolic state in the HFD/L-NAME model and identify ketone supplementation as a partial metabolic rescue strategy in this context. At the same time, they highlight an important limitation of murine HFpEF models: such models do not faithfully reproduce the metabolic phenotype of human HFpEF and should therefore be interpreted as experimental systems rather than human disease equivalents.

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.

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.

AimsHypertrophic cardiomyopathy (HCM) is a genetic heart disease that leads to left ventricular hypertrophy, heart failure, and arrhythmias. Pathogenic missense variants in the gene myosin binding protein C (MYBPC3) cluster within its internal subdomains C3 and C6. The protein, myosin binding protein C (MyBP-C), expressing these variants, normally localizes to the myofilaments, leaving uncertainty regarding the mechanism(s) by which they cause HCM. MethodsWe probed the mechanisms of these variants by analyzing (1) their prevalence in an international registry of patients with HCM, (2) total MyBP-C levels and the allelic fraction of mutant MyBP-C in human left ventricular myectomy heart tissue, and (3) performing flag-immunoprecipitation and proximity labeling mass spectrometry of wild-type MyBP-C and four pathogenic missense variants (Arg495Gln, Arg502Trp-C3 subdomain, Trp792Arg, Arg810His-C6 subdomain) to determine the change in MyBP-C interacting and proximity proteins induced by these variants. ResultsWe found that in patients with HCM who had any MYBPC3 pathogenic variant, 17.9% of them had a missense variant localized to the C3 or C6 subdomain. Unlike truncating variants, missense variants did not reduce MyBP-C content relative to myosin. The mutant allelic fraction of MyBP-C varied from 10-67.0% across samples. Flag-immunoprecipitation mass spectrometry identified 252 MyBP-C interacting proteins. Pathogenic missense variants disrupted 23 MyBP-C protein interactions, including lysosomal Ragulator-Rag complex proteins (Rraga, Rragc, LAMTOR4). Proximity labeling mass spectrometry was more sensitive, identifying 3,240 MyBP-C proximity proteins. Pathogenic missense variant (s) altered proximity of 789 proteins (69.4% increased and 30.5% decreased relative to wild-type MyBP-C). Proteins that were increased in proximity to missense MyBP-C were enriched for proteins within thin filament. ConclusionPathogenic MYBPC3 missense variants within the C3 and C6 subdomains are present in a substantial subset of patients with HCM. Our findings implicate unique mechanisms of these variants distinct from haploinsufficiency, potentially driven in part by enhanced protein-protein interactions with the thin filament within the sarcomere. Translational perspectivePathogenic missense variants in the gene myosin binding protein C (MYBPC3) cause hypertrophic cardiomyopathy via an unknown mechanism. These variants exhibit normal cellular localization and stability. We have demonstrated that these variants are present in 17.9% of patients with HCM and a pathogenic MYBPC3 variant and do not reduce the level of total MyBP-C, the protein encoded by MYBPC3, within human left ventricular tissue. Using Flag-immunoprecipitation and proximity labeling mass spectrometry, we identified differential interacting proteins and proximity proteins, respectively, with proximity labeling mass spectrometry displaying much higher sensitivity. This was particularly true for the detection of potential gain-of-function shifts in MyBP-C environment within the sarcomere. Three of the four pathogenic missense proteins evaluated demonstrated increased proximity to thin filament proteins. This may suggest that particular missense variants within C3 and C6 internal subdomains result in a conformational change that favors thin filament binding and activation. These findings have important implications for the development of sarcomeric modulators that address the underlying mechanism HCM caused by MYBPC3 missense variants.

BACKGROUNDSmooth muscle cells (SMCs) comprise the majority of cells in human atherosclerotic lesions and are thought to be a major source of cholesterol-overloaded foam cells in human and mouse atheromas. However, the transcriptomic profile, specific markers, and biologic itinerary of SMC foam cells relative to macrophage foam cells remain poorly defined. METHODSSingle-cell RNA sequencing (scRNA-seq) was performed on fresh coronary artery segments from heart transplant recipients with early- to intermediate-stage atherosclerosis. Gene expression in a putative SMC foam cell cluster was compared with cultured SMCs loaded with aggregated low-density lipoprotein (agLDL) or cholesterol-methyl-{beta}-cyclodextrin (Chol-M{beta}CD). Candidate markers distinguishing SMC from macrophage foam cells were validated using additional publicly-available scRNA-seq datasets, Xenium spatial transcriptomics, and immunofluorescence microscopy of human coronary atheromas. Pathway analysis was performed using Gene Set Enrichment Analysis Hallmark gene sets. RESULTSA distinct SMC foam cell cluster derived from fibromyocytes ("lipomyocytes") was identified using markers induced by in vitro cholesterol loading. agLDL loading reproduced the lipomyocyte transcriptional profile, whereas Chol-M{beta}CD induced an inflammatory phenotype colocalizing with macrophages rather than lipomyocytes. Lipomyocytes highly expressed SERPINE1, encoding plasminogen activator inhibitor-1 (PAI-1), and CFH, encoding complement factor H, which were validated in human coronary lesions by spatial transcriptomics and immunofluorescence microscopy. Compared with macrophage foam cells, lipomyocytes demonstrated distinct pathway activation, including enrichment of extracellular matrix, coagulation and angiogenesis pathways. CONCLUSIONSSMC foam cells, or lipomyocytes, represent a distinct foam cell phenotype with unique markers and biologic programs that differ from macrophage foam cells during atherosclerotic plaque development. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LISmooth muscle cell (SMC) foam cells, or lipomyocytes, arise from fibromyocytes and exhibit a transcriptomic profile that is markedly distinct from that of macrophage foam cells. C_LIO_LIIn vitro loading of SMCs with aggregated LDL recapitulates the gene expression profile of SMC foam cells in human coronary atheromas, whereas loading with cyclodextrin-bound cholesterol does not. C_LIO_LIPlasminogen activator inhibitor 1 (PAI-1, encoded by SERPINE1) and Complement Factor H are specific markers of SMC foam cells and are not expressed by macrophage foam cells. C_LI What Are the Clinical Implications?O_LISMCs contribute a substantial proportion, and potentially the majority, of foam cells in atherosclerotic lesions. C_LIO_LIDefining the biological trajectory of SMC foam cells within plaques is critical for understanding their roles in plaque progression, rupture and thrombosis, and for establishing their relevance as a distinct therapeutic target to reduce major cardiovascular events. C_LI

Background: Thoracic aortopathy is a disorder with genetic influence usually presenting in adulthood for which family screening is potentially desirable. Family screening is recommended, but the predictors of a positive screen and real-world pickup rates are unknown. Methods: This was a retrospective cohort of 1022 probands (first affected family member identified) with thoracic aortopathy and one or more features suggestive of a genetic etiology, and their presenting family members, assessed in a cardiac clinic (2009?2024). Imaging and genetic testing were employed in family screening. The prespecified outcomes were uptake and pickup rate of family screening, and proband and family member specific characteristics that predicted a positive family screen. Results: Among probands, 43.5% had one or more family member screened, with an average of 3 relatives per successful proband. 27.6% of family members screened positive. A pre-existing family history of aortopathy was the only variable predicting a higher incidence rate for positive family screen (p = 0.0003). Age of presentation < 60 was not predictive. For family members, extravascular features (p < 0.0001), closer relation to the proband (p < 0.02), male sex (p < 0.0001) and older age (p< 0.0001) all predicted a positive screen. Family members were eight times more likely to screen positive through imaging as compared to genetic testing. Probands with a genetic diagnosis of Marfan and Loeys Dietz syndromes accounted for only 4% of the total. Conclusions: Proband-initiated family screening for thoracic aortopathy has a high yield of affected individuals, even among older probands.

BackgroundSpontaneous coronary artery dissection (SCAD) is a well-recognised cause of acute coronary syndrome particularly among women without conventional cardiovascular risk factors. Increasing evidence indicates a genetic contribution; however, the underlying genetic architecture of SCAD remains insufficiently understood. ObjectiveThe aim of this study was to assess the prevalence of rare variants in previously reported SCAD associated genes and to explore the potential presence of novel genetic alterations in well-characterised Swedish patients with SCAD. MethodsThe study comprised 201 patients enrolled in SweSCAD, a national project examining the clinical characteristics, aetiology, and outcomes of SCAD. All individuals had a confirmed diagnosis based on invasive coronary angiography. Comprehensive exome sequencing was performed to identify rare variants contributing to disease susceptibility. ResultsGenetic variants that have been associated with SCAD according to current clinical genetics practice for variant reporting were identified in approximately 4 % of patients. In addition, rare potentially relevant variants were detected in almost 60 % of patients in genes associated with vascular integrity and vascular remodelling. ConclusionThis study supports SCAD as a genetically complex arteriopathy, driven by rare high-impact variants together with broader polygenic susceptibility. Variants in collagen, vascular extracellular matrix, and oestrogen-responsive pathways provide biologically plausible links to female-predominant disease. Although the diagnostic yield of clearly actionable variants is modest, these findings support broader genomic evaluation beyond overt syndromic presentations and highlight the need for larger integrative genomic and functional studies to refine risk stratification and management.

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

Abdominal aortic aneurysm (AAA) is a chronic inflammatory vascular disease characterized by progressive extracellular matrix degradation, vascular smooth muscle cell (VSMC) loss, and immune cell infiltration, ultimately leading to aortic dilation and rupture. Although vitamin 25(OH)D3 deficiency has been associated with cardiovascular inflammation, its mechanistic role in AAA pathogenesis remains poorly defined. Here, we investigated the role of vitamin D{square} mediated signaling to regulate complement pathway activation, particularly the C3a axis, to modulate aneurysm development. Single cell-RNA sequencing analysis of human tissue demonstrated significant differences in Vitamin D and complement pathway-related genes in VSMCs in AAAs compared to control aortic tissue. Using a murine elastase-induced AAA model, we observed that vitamin D3-deficient diet significantly enhances aortic dilation, leukocyte infiltration, proinflammatory cytokine expression and elastin fragmentation, as well as decreases SMC -actin expression compared with vitamin D3-sufficient conditions. Furthermore, vitamin D3 deficiency was accompanied by increased aortic expression of complement component C3a that correlated with vascular inflammation and remodeling during AAA progression. Pharmacological blockade with a C3a receptor antagonist (C3aRA) markedly attenuated AAA formation in two established murine AAA models with concomitant reductions in proinflammatory cytokines and preservation of aortic wall structure. In vitro studies demonstrated that stimulation of VSMCs significantly increased C3a production, which was suppressed by calcitriol (active form of Vitamin D) treatment. These studies suggest that the vitamin D-C3a axis is a critical regulator of vascular inflammation and AAA progression, and postulate that restoring vitamin D{square} sufficiency or targeting C3a signaling may represent a novel therapeutic strategy to limit AAA growth and rupture. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/730431v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@18d5d8forg.highwire.dtl.DTLVardef@1f98952org.highwire.dtl.DTLVardef@1a66a95org.highwire.dtl.DTLVardef@9f6439_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: Coronary artery calcium (CAC) scores inform subclinical atherosclerotic cardiovascular disease (ASCVD) burden, helping guide preventative treatments. However, prediction of cardiovascular (CV) events by CAC is largely limited to ASCVD outcomes. This study investigated whether a previously validated proteomic test for predicting a broad composite of four-year CV events could enhance the prognostic utility of CAC. Methods: We used a 27-protein CV risk score (Prot-CVR), derived from ~5,000 SomaScan? Assay plasma protein measurements, to predict four-year risk of a composite CV and mortality outcome (myocardial infarction, stroke/TIA, heart failure hospitalization, death) in 2,122 participants with ?1 CV risk factors from the Multi-Ethnic Study of Atherosclerosis (MESA) observational cohort at exam 5 and compared predictions to CAC Agatston scores. Discriminatory performance was assessed using C-Index and 4-year area under the curve (AUC). Cox Proportional Hazard (CoxPH) ratios were calculated for the composite outcome, ASCVD outcome (myocardial infarction, resuscitated cardiac arrest, stroke, coronary heart disease death), and individual events. Changes in Prot-CVR and CAC scores from baseline to MESA exam 5 (+10-years) in CV event versus event-free participants were assessed using 2-tailed paired t-tests. CoxPH regression models of CV event status distributed by Prot-CVR, CAC, and relevant co-variates were evaluated for performance relative to individual models. Results: Individual Prot-CVR and CAC models predicting the composite outcome had comparable 4-year AUCs, but Prot-CVR had a higher C-index (0.68 (0.65-0.70) versus 0.63 (0.60-0.65), p=0.001) and greater hazard ratios for the composite outcome (p<0.001), death (p<0.001), and heart failure (p=0.015). A combined CoxPH model of Prot-CVR + CAC + Age had a higher 4-year AUC (0.72, p<0.05) and C-Index (0.71, p<0.05) than Prot-CVR or CAC alone. Both Prot-CVR and CAC scores detected an increase in risk prior to an approaching CV event in ~10-year sensitivity-to-change analysis. For 49.6% of MESA population with CAC=0 at baseline, Prot-CVR was greater in composite event versus event free participants at 4 years (0.23 versus 0.15, p=0.006) and full follow-up (0.18 versus 0.13, p<0.001). Conclusion: Protein testing complements CAC for CV risk assessment although the improvement is modest. Prot-CVR may resolve which patients with CAC=0 are at heightened CV risk.

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

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.

Objective: To compare RNA-sequencing-derived transcriptomic profiles of thoracic aortic aneurysm tissue from individuals with bicuspid versus trileaflet aortic valves. Methods: Human ascending aortic tissue was collected from patients undergoing cardiac surgery at a single institution between January 2021 and December 2022 with bicuspid aortic valves (BAV) and trileaflet aortic valves (TAV) with (-A) and without (-N) thoracic aortic aneurysm. TAV-N tissue was collected from heart transplant donors. The decision to perform ascending aortic replacement was at surgeon discretion following ACC/AHA guidelines. Bulk RNA was extracted from the aortic wall, and Illumina RNA Sequencing performed. Differential gene expression analysis, enrichment analyses, network analysis, and deconvolution single cell-mapping were performed in R. Cell-type specificity of differentially expressed genes was determined using an established Aorta single cell RNA sequencing matrix. Results: Tissue samples from 60 patients were included: 4 TAV-N, 16 BAV-N, 28 BAV-A, and 12 TAV-A. Average absolute aortic diameter was 5.1 +/- 0.38 cm for BAV-A and 5.3 +/- 0.44 cm for TAV-A, as measured on pre-operative CT. Gene ontology analyses of differentially expressed genes revealed enrichment of genes associated with extracellular matrix (ECM) organization, cellular receptor interactions and vascular smooth muscle cell (VSMC) function in BAV-A and BAV-N. In contrast, analysis of TAV-A versus TAV-N showed enrichment in genes associated with immune and inflammatory processes. Cell-type specificity analysis revealed a downregulation of genes associated with ECM components, cell signaling, and ECM remodeling in mesenchymal cells, VSMCs, and matrix fibroblasts specifically in BAV-A versus BAV-N. Conclusions: The transcriptome changes observed in aneurysmal aortas of BAV and TAV patients are distinct, suggesting mechanistic differences contributing to aneurysm development and progression. The observed differences in gene expression between the non-aneurysmal aortas may signify a predisposition to aneurysm development unique to BAV aortopathy.

Background: Heart failure (HF) and body mass index (BMI) share substantial genetic architecture, which may lead genetically informed target discovery to preferentially identify adiposity-related pathways. We sought to identify circulating proteins associated with HF beyond this shared genetic component. Methods: We applied GWAS-by-subtraction to overall HF, nonischemic HF, and nonischemic HF with reduced or preserved ejection fraction to derive BMI-related and BMI-subtracted HF components. We then performed proteome-wide cis-pQTL Mendelian randomization and colocalization using four independent proteomic cohorts, followed by tissue-specific eQTL colocalization, cardiac transcriptomic annotation, and druggability assessment. Results: Compared with the original HF phenotypes, the BMI-subtracted components showed attenuated genetic correlations with BMI (0.045-0.147) while retaining 28 independent loci for overall HF and nine for nonischemic HF. Across 19,930 protein-HF tests, 11 associations involving nine proteins were prioritized by the Mendelian randomization and colocalization analyses. For example, a 1-SD increase in genetically predicted CELSR2 abundance was associated with lower overall HF risk (odds ratio, 0.96 [95% CI, 0.94-0.98]; P=8.6x10-7), whereas a 1-SD increase in genetically predicted CSF3 abundance was associated with higher nonischemic HF risk (odds ratio, 1.32 [95% CI, 1.18-1.48]; P=2.0x10-6). CELSR2 and TMEM106B colocalized with cis-eQTLs in failing left ventricular myocardium, and DAG1 showed cardiomyocyte enrichment with concordant downregulation in failing hearts. Conclusions: We identified nine circulating proteins associated with HF beyond the genetic component shared with BMI. These findings extend the range of genetically supported pathways implicated in HF and nominate candidate proteins for further mechanistic and therapeutic investigation.