Nature Cardiovascular Research

Neurological complications frequently impact morbidity, mortality, and quality of life in patients with cardiovascular disease, yet the biological mediators connecting cardiovascular and neurological disease are poorly understood. Leveraging data from 53,014 individuals with plasma proteomic profiles and 50,228 with cardiac and brain MRI from the UK Biobank, we systematically identified circulating proteins correlated with MRI imaging-derived phenotypes (IDPs) (404 proteins with cardiac IDPs; 76 with brain IDPs; 37 with both). Identified proteins were remarkably enriched for biomarkers and mediators of disease in one or both organs. Expression analyses suggested these proteins largely originate from fibroblasts, smooth muscle cells, and macrophages in the arterial vasculature. Pathway analyses highlighted cytokine and vasculature-related processes for cardiac IDPs-associated proteins and extracellular matrix pathways in brain IDPs-associated proteins. Mendelian Randomization and genetic co-localization supported causal roles for most (>63%) of the proteins in disease pathogenesis in one or both organs. Over 90% of the implicated candidates have not previously been established as clinical biomarkers or therapeutic targets. These studies underscore the value of large-scale integrated multi-organ datasets, including plasma proteomics, imaging-derived endophenotypes, and genetics, in unraveling complex disease pathobiology, highlight the close connections between heart and brain disease, and provide a catalog of hundreds of novel candidate biomarkers and therapeutic targets.

BackgroundAscending thoracic aortic dissection (ATAD) is characterized by extensive macrophage (M{Phi}) accumulation and profound inflammation; however, the mechanisms sustaining pro-inflammatory M{Phi} activation remain incompletely defined. Emerging evidence indicates that epigenetically generated immune memory drives innate immune cells toward persistent inflammatory states. In this study, we investigated whether epigenetic reprogramming governs M{Phi} phenotypic fate and contributes to ATAD pathogenesis. MethodsWe performed single-cell RNA sequencing of human ascending aortic tissues from controls, patients with ascending thoracic aortic aneurysm (ATAA), and patients with acute ascending thoracic aortic dissection (ATAD). We also performed integrated single-cell RNA sequencing, single-cell ATAC sequencing, and spatial transcriptomics in an angiotensin II (Ang II)-infused mouse model. The role of the STING-IRF3 signaling axis in M{Phi} epigenetic programming was examined using M{Phi}-Sting -/- and M{Phi}-Irf3-/- mice. ResultsIn human and mouse aortic tissues, we identified multiple functional M{Phi} populations including pro-inflammatory, phagocytic/anti-inflammatory, proliferative, and reparative/healing M{Phi}s. Aortic M{Phi}s in both sporadic ATAD patients and Ang II-induced ATAD mice exhibited a pronounced pro-inflammatory bias with enhanced differentiation toward pro-inflammatory M{Phi}s and impaired differentiation toward phagocytic/anti-inflammatory states. Pro-inflammatory M{Phi}s were particularly abundant in dissection sites, whereas phagocytic M{Phi}s were enriched in discrete adventitial niches. Origin analyses revealed a substantial increase in CCR2 recruited M{Phi}s within the aortic wall, which preferentially differentiated into pro-inflammatory M{Phi}s. In contrast, LYVE1 resident M{Phi}s-- predominantly biased toward phagocytic phenotypes--were markedly depleted in ATAD. Single-cell ATAC sequencing identified coordinated chromatin remodeling with increased accessibility at pro-inflammatory gene loci and decreased accessibility at phagocytic gene loci. Among candidate transcriptional regulators identified, IRF family TFs, including IRF3 emerged as unique factors capable of simultaneously promoting pro-inflammatory gene programs while suppressing phagocytic gene expression. Mechanistically, STING-IRF3 signaling orchestrates this biased transcriptional state, likely through coordinated BRG1-dependent chromatin opening at pro-inflammatory gene loci and chromatin closing at phagocytic/anti-inflammatory gene loci. M{Phi} specific Sting -/- and Irf3-/- mice exhibited attenuated inflammatory reprogramming and reduced aortic destruction and dissection. ConclusionsThese findings identify STING-IRF3-mediated epigenetic programming of M{Phi}s as a fundamental mechanism driving aortic inflammation and ATAD development. Targeting M{Phi} epigenetic programming may represent a promising therapeutic strategy to prevent aortic dissection. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/701198v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@c97bcdorg.highwire.dtl.DTLVardef@1df0ca8org.highwire.dtl.DTLVardef@b7fd04org.highwire.dtl.DTLVardef@1443e16_HPS_FORMAT_FIGEXP M_FIG C_FIG

Direct identification of physically interacting cells in vivo remains challenging because conventional interactome analyses infer signaling partners from transcriptomes and cannot reveal which cells are in direct contact. In pressure-overload induced cardiac remodeling, VEGF-A plays a central role in the maintenance of vascular integrity and cardiac function. However, the cell type which produces VEGF-A and how the VEGF-A peptide is delivered to vascular endothelial cells remains unclear. Here, we developed a genetically encoded platform that combines REFLEX mice with HUNTERuni-seq, enabling unbiased detection and transcriptional profiling of the cells that physically interact with vascular endothelial cells. The REFLEX and HUNTERuni-seq approach identified subpopulations of Vegfa positive macrophages which we named perivascular angiogenic macrophages (PVAMs). Although the amount of VEGF-A in PVAMs is small, loss of VEGF-A in PVAMs impaired angiogenesis and systolic function during pressure overload. We additionally show that direct contact between PVAMs and endothelial cells is critical for the delivery of VEGF-A to endothelial cells. Conventional interactome analysis predicted that cardiomyocytes as dominant sources of VEGF-A in the heart. However, cardiomyocyte Vegfa deletion had no effect on capillary density nor systolic function in a model of heart failure. These results suggest that VEGF-A signaling does not rely on free diffusion through the interstitium and that cellular proximity and physical contact between PVAMs and endothelial cells are the key determinants of effective signal delivery. Together, these findings establish REFLEX and HUNTERuni-seq as a versatile platform for uncovering biologically critical cell-to-cell interactions and provide new insight into intercellular communication in pathological tissue contexts.

Glucagon-like peptide-1 receptor (GLP1R) agonists improve glycaemic control, induce weight loss, and consistently reduce major adverse cardiovascular events. However, the mechanistic basis of their cardioprotective effects remains incompletely understood, particularly whether benefits arise solely from systemic actions or also involve direct cardiac GLP1R signalling. To address this, we performed integrated single-cell and single-nucleus transcriptomics to map GLP1R gene expression across human and murine organs, cardiac cell types, disease states, and hiPSC-derived cardiac organoids. In humans, GLP1R expression was predominantly pancreatic, with low cardiac expression largely restricted to cardiomyocytes and consistently upregulated across ischaemic, dilated, and hypertrophic cardiomyopathy. In contrast, murine cardiac Glp1r expression was confined to endocardial cells and remained unchanged in heart disease. Other cardiac cell types, including fibroblasts, endothelial cells, and mural cells, showed minimal GLP1R expression in both species. Human cardiac organoids recapitulated ventricular GLP1R patterns closer to adult human myocardium than murine tissue. Together, these findings indicate that GLP1R is primarily extracardiac but selectively induced in failing human myocardium, supporting a model in which myocardial GLP1R signalling augments systemic mechanisms to confer GLP1R agonist-mediated cardioprotection.

Genetic cardiomyopathies consist of a heterogeneous group of myocardial disorders caused by variants that disrupt key regulators of cardiac structure and function. Variants in PLN, encoding phospholamban (PLN), the main inhibitor of the sarco/endoplasmic reticulum Ca{superscript 2}-ATPase 2a (SERCA2a), have been linked to both dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM). Among these, the PLN Arg14del (R14del) variant is the most prevalent. PLN R14del cardiomyopathy is characterized by the accumulation of large perinuclear PLN aggregates in cardiomyocytes of end-stage heart failure tissue. However, the mechanisms driving PLN aggregate formation and their role in disease progression remain unresolved. Using a humanized plna R14del zebrafish model, left ventricular tissue from end-stage PLN R14del cardiomyopathy patients and pharmacological modeling in wild type (WT) cardiac slices, we demonstrate that previously described PLN aggregates represent accumulated sarcoplasmic reticulum (SR)-derived PLN-containing vesicles that form due to impaired SERCA2a activity and increased cytosolic Ca{superscript 2} levels. Furthermore, these SR-derived vesicles often localize adjacent to lysosomes. Interestingly, Ca2+ dysregulation in plna R14del hearts leads to reduced lysosomal function, resulting in SR-derived vesicle accumulation at the microtubule organizing center (MTOC). This perinuclear accumulation induces microtubule aster formation and subsequent cellular disorganization, including sarcomere misalignment and nuclear deformation. Strikingly, reactivation of lysosomal function through fasting reduces SR-derived vesicle accumulation, restores microtubule integrity, and rescues cellular organization in plna R14del zebrafish hearts. Together, these findings identify impaired lysosomal clearance of SR-derived vesicles and the resulting microtubule disorganization as key pathological mechanisms driving PLN R14del cardiomyopathy. Additionally, our results highlight lysosomal reactivation as a promising potential therapeutic strategy to halt or reverse PLN R14del cardiomyopathy progression. Main findingsO_LIPLN aggregates in PLN R14del cardiomyopathy represent SR-derived vesicles formed due to Ca{superscript 2} dysregulation. C_LIO_LIThese SR-derived vesicles often localize perinuclearly at the microtubule organizing center (MTOC), where they are positioned adjacent to lysosomes. C_LIO_LICa2+ dysregulation leads to lysosomal dysfunction which drives vesicle accumulation responsible for microtubule remodeling and pathological cellular rearrangements. C_LIO_LILysosomal reactivation restores vesicle clearance and rescues cardiomyocyte structure. C_LI

BackgroundHeart failure, arrhythmia, and sudden cardiac death are common outcomes of cardiomyopathies, which are molecularly diverse heart muscle disorders marked by structural and functional myocardial dysfunction. The lack of sensitive molecular biomarkers that precede overt physiological deterioration makes early diagnosis difficult despite advancements in imaging and clinical classification. The immune transcriptional landscape across cardiomyopathy subtypes is still poorly understood, despite growing evidence linking both innate and adaptive immune dysregulation, such as macrophage activation and T-cell and inflammatory cytokine networks, as active contributors to myocardial remodelling and disease progression. MethodsWe performed a multi-cohort integrative transcriptomic analysis of 1,068 cardiac tissue samples from five publicly available GEO datasets (GSE57338, GSE5406, GSE36961, GSE141910, GSE47495) spanning dilated, ischemic, hypertrophic, and peripartum cardiomyopathy. Using a fully scripted R and Python pipeline, we conducted differential expression analysis (limma), immune cell deconvolution (xCell), pathway enrichment (clusterProfiler), weighted gene co-expression network analysis (WGCNA), and regularised machine learning classification (LASSO, Random Forest). Cross-dataset validation was performed in two independent cohorts on different microarray platforms. ResultsDifferential expression analysis identified 43 primary DEGs (FDR < 0.05, |log2FC| > 1.0), revealing a coherent immune-fibrotic program characterized by loss of anti-inflammatory macrophage markers (CD163, VSIG4), complement dysregulation (FCN3), innate interferon activation (IFI44L, IFIT2), and ECM remodelling (ASPN, SFRP4, LUM). xCell deconvolution identified coordinated depletion of adaptive immune populations in failing myocardium. WGCNA defined a fibrosis hub module (brown; CTSK, SULF1, SFRP4) and an immune collapse module (turquoise; MYD88, TNFRSF1A, LAPTM5). A nine-gene LASSO classifier achieved a cross-validated AUC of 0.986, with HMOX2 as the top-discriminating feature, implicating ferroptosis in cardiomyocyte death. Cross-platform validation in an independent HCM cohort (GSE36961) demonstrated a directional concordance of 34.9%. ConclusionsThis study defines a reproducible immune-fibrotic transcriptional signature of human cardiomyopathy, nominates HMOX2 and ferroptosis as central pathomechanisms, and provides a validated nine-gene biomarker panel for future translational investigation.

Mitral valve prolapse (MVP) is the most common cause of primary mitral regurgitation and is associated with the development of malignant arrhythmias, often in the context of myocardial fibrosis. The genetic architecture of MVP, and whether there are genetic factors explaining why only some individuals with MVP have adverse outcomes, remains poorly understood. We performed a meta-analysis of genome-wide association studies (GWAS) for MVP encompassing 21,517 cases among a total sample size of over 2.2 million individuals. We discovered 89 genomic risk loci for MVP, of which 72 were novel findings. Prioritization of causal genes and pathways using epigenetic and transcriptomic data from mitral valve and extra-valvular tissues replicated known gene associations to MVP including those involved in TGF-{beta} signaling and extracellular matrix biology, but additionally emphasized a role in MVP for biological pathways relevant to cardiomyocyte biology. Accordingly, we identified several MVP risk loci with pleiotropy to cardiomyopathies, especially hypertrophic cardiomyopathy, and demonstrated a significant genetic correlation between MVP and hypertrophic cardiomyopathy. Finally, we interrogated snRNA-seq data in human papillary muscle tissue from two individuals with severe MVP, characterizing genes associated with both risk of papillary muscle fibrosis and MVP.

Excessive innate immune activation drives adverse remodeling after myocardial infarction (MI), yet the upstream mechanisms by which macrophages sense ischemic danger signals remain poorly defined. Here we tested whether macropinocytosis functions as a mediator of post-ischemic inflammation and whether the Na/H exchanger SLC9A1 links membrane ion transport to innate immune activation in the injured heart. Macropinocytosis was robustly activated in infarct-associated macrophages, which are the predominant cell type with the macropinocytotic activity in the injured heart. Pharmacologic inhibition of macropinocytosis with 5-(N-ethyl-N-isopropyl)amiloride (EIPA) improved cardiac function and attenuated post-MI remodeling. EIPA also attenuated cardiac inflammatory responses induced by systemic lipopolysaccharide and Poly(I:C). To define macrophage-intrinsic mechanisms, we generated monocyte- and monocyte-derived macrophage-specific Slc9a1 knockout mice. Genetic deletion of Slc9a1 recapitulated the cardioprotective effects of EIPA and markedly suppressed interferon-stimulated gene programs in infarct-associated macrophages, as revealed by single-cell RNA sequencing. Mechanistically, SLC9A1 promoted endocytic uptake of Poly(I:C) acid and enhanced endosome-dependent inflammatory signaling. Together, these findings identify macrophage macropinocytosis as a regulator of innate immune activation after MI and reveal SLC9A1 as a previously unrecognized link between membrane ion transport and inflammatory signaling in the injured heart. Targeting SLC9A1-dependent membrane trafficking pathways may therefore represent a strategy to limit maladaptive inflammation in ischemic heart disease.

Cardiac fibrosis driven by persistent myofibroblast activation is a major contributor to adverse ventricular remodeling and heart failure. Bromodomain and extra-terminal domain (BET) inhibition reduces fibrosis and hypertrophy in preclinical models, but direct targeting of the BET co-activator BRD4 is limited by family homology and potential systemic toxicity. Sertad4 (SERTA domain containing protein 4) is a BRD4-dependent gene induced in activated cardiac fibroblasts, yet its role in cardiac pathology is unknown. Here, we examined Sertad4 expression and function in human heart failure and in murine myocardial infarction (MI). SERTAD4 protein was increased in left ventricular tissue from heart failure patients compared with non-failing controls. In Sertad4/LacZ reporter mice, MI triggered strong Sertad4 activation localized to the infarct scar and border zone, with minimal expression in remote myocardium; single-nucleus RNA sequencing further demonstrated that Sertad4 expression is predominantly fibroblast-restricted and significantly upregulated after MI. To test causality, we subjected global Sertad4 knockout mice to 28-day left anterior descending coronary artery ligation. Sertad4 deletion attenuated post-MI remodeling, reduced hypertrophy and ventricular dilation, and preserved systolic function. Consistent with improved structure and function, knockout hearts exhibited reduced cardiomyocyte cross-sectional area and decreased expression of fibrosis and hypertrophy associated genes. Together, these findings identify Sertad4 as a fibroblast enriched regulator of pathological remodeling and suggest that targeting Sertad4 may offer a more cell type-selective alternative to direct BET/BRD4 inhibition for limiting cardiac fibrosis and progression to heart failure

BackgroundPathogenic desmin (DES) variants have been implicated in early-onset atrial disease, yet the mechanisms by which desmin dysfunction alters atrial structure and function remain unclear. Desmin anchors the cytoskeleton to the nuclear envelope (NE) through the linker of nucleoskeleton and cytoskeleton (LINC) complex, suggesting that defects in this network may drive atrial cardiomyopathy. MethodsHuman desmin wild-type (WT) and the pathogenic variants p.S13F, p.N342D, and p.R454W were stably expressed in HL-1 atrial cardiomyocytes. Desmin organization, nuclear morphology, LINC-complex integrity (nesprin-3, lamin A/C), and DNA leakage, assessed by cyclic GMP-AMP synthase (cGAS), were analyzed by confocal microscopy. Action potential duration (APD) and calcium transients (CaT) were measured optically. Human myocardium samples from DES variant carriers were analyzed for validation. Data-independent acquisition (DIA) mass spectrometry profiled atrial proteomes from desmin-network (DN) and titin variant carriers and controls. The heat-shock proteins (HSPs) inducer geranylgeranylacetone (GGA) was evaluated for rescue effects. Resultsp.N342D caused severe filament-assembly defects with prominent perinuclear aggregates, whereas p.S13F showed mixed phenotypes with frequent perinuclear aggregates, and p.R454W largely preserved filamentous networks. p.N342D and p.S13F induced nuclear deformation with disrupted nesprin-3 and lamin A/C distribution. In p.N342D and p.S13F, desmin aggregates drove focal lamin A/C accumulation, nuclear envelope (NE) rupture, DNA leakage, and increased cGAS activation. DES variants significantly shortened APD20/90 and reduced CaT amplitude, indicating pro-arrhythmic electrical remodeling. Atrial proteomics revealed a DN-specific signature enriched for cytoskeletal, NE, intermediate filament, and chaperone pathways, consistent with the structural injury observed in vitro. GGA prevented desmin aggregation and nuclear morphology changes, and mitigated APD shortening in p.N342D-expressing cardiomyocytes. Human myocardium from DES variant carriers showed concordant desmin aggregation and polarized lamin A/C distribution. ConclusionsDES variants induce a desmin-dependent atrial cardiomyopathy characterized by cytoskeletal disorganization, disruption of LINC-complex, NE rupture with DNA leakage, and pro-arrhythmic electrophysiological remodeling. These findings provide mechanistic insight into how DN variants promote atrial disease. HSPs induction by GGA partially restores structural and functional integrity, identifying a potential therapeutic approach for desmin-related atrial cardiomyopathy. Clinical perspectiveWhat is new? O_LIPathogenic DES variants induce a previously unrecognized atrial cardiomyopathy characterized by desmin aggregation, and desmin-network (DN) collapse, disruption of the linker of nucleoskeleton and cytoskeleton (LINC) complex, and nuclear envelope rupture with DNA leakage. C_LIO_LIVariants that lead to desmin aggregation (e.g., p.N342D) cause focal lamin A/C polarization, cyclic GMP-AMP synthase (cGAS) activation, and structural injury at the nuclear envelope. C_LIO_LIDES variants produce pro-arrhythmic electrical remodeling, including action potential duration shortening and impaired Ca{superscript 2} handling in HL-1 atrial cardiomyocytes. C_LIO_LIAtrial proteomics from DN variant carriers reveals enrichment of pathways related to cytoskeletal, nuclear envelope, intermediate filament, and chaperone, supporting a desmin-dependent remodeling program. C_LIO_LIThe heat-shock protein inducer geranylgeranylacetone (GGA) prevents desmin aggregation, restores nuclear morphology, and mitigates electrical and Ca{superscript 2} handling remodeling. C_LI What are the clinical implications? O_LIThese findings establish DN dysfunction as a distinct cause of atrial cardiomyopathy, providing a mechanistic basis for the association between pathogenic DES variants and atrial arrhythmias, including atrial fibrillation. C_LIO_LINuclear envelope rupture and cytosolic DNA leakage represent new mechanistic evidence which links cytoskeletal injury and atrial arrhythmogenesis. C_LIO_LIIdentifying structural vulnerability in DES variant carriers fosters awareness of genetic counseling for atrial disease, enabling early detection and risk stratification. C_LIO_LIThe protective effects of GGA suggest that restoring proteostasis may be a therapeutic strategy for desmin-related atrial cardiomyopathy and potentially other genetic atrial diseases. C_LI Novelty and significance statementO_ST_ABSNoveltyC_ST_ABSThis study identifies a desmin-dependent atrial cardiomyopathy driven by cytoskeletal aggregation, LINC-complex disruption, and nuclear envelope rupture with DNA leakage. We show that pathogenic DES variants are associated with pro-arrhythmic molecular remodeling and that human atrial proteomics confirm nuclear envelope and cytoskeletal injury as core features. Importantly, the heat-shock protein-inducer GGA rescues structural, molecular, and electrophysiological defects, revealing a modifiable pathway in desmin-mediated atrial disease. SignificanceThese findings provide the first integrated mechanistic explanation linking DN variants to atrial cardiomyopathy. By uncovering nuclear envelope rupture and cGAS activation as key drivers of atrial cardiomyopathy, this work expands the molecular framework for inherited atrial disease and highlights proteostasis enhancement as a potential therapeutic strategy for patients carrying DES and related cytoskeletal variants. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/26348559v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1fb0bfborg.highwire.dtl.DTLVardef@cfc00borg.highwire.dtl.DTLVardef@1493578org.highwire.dtl.DTLVardef@1556b61_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sex differences in atherosclerotic plaque biology underlie clinically distinct manifestations of acute coronary syndromes, yet the molecular mechanisms driving these differences remain incompletely understood. Endothelial-to-mesenchymal transition (EndMT) is increasingly recognized as a contributor to plaque remodeling, but whether EndMT is regulated in a sex-specific and stage-dependent manner across atherosclerosis has not been systematically examined. Here, we integrated bulk RNA sequencing with single-cell transcriptomic analyses in endothelial cells and human atherosclerotic plaques to define sex-specific EndMT regulation across healthy and advanced-stage disease. Using trajectory analyses in endothelial cells undergoing EndMT, we observed pronounced sex-specific regulation in healthy endothelium, with females exhibiting stronger early EndMT activation, whereas endothelial cells derived from atherosclerotic plaques displayed markedly attenuated sex differences with disease progression. Consistent sex-divergent pseudotime trajectories were observed in human carotid plaque endothelial single-cell RNA-seq data, with females showing greater EndMT activation at earlier stages and males at later stages. Together, these findings support a stage-dependent model of sex-specific EndMT regulation, indicating that the functional consequences of EndMT are highly context dependent and may differ across early and late disease stages. Integration of these datasets prioritized high-confidence sex-specific EndMT regulators, including COL4A1, PECAM1, CD151, JAG1, FN1, NEDD9, PODXL, MAFB, PROCR, and CDH13, providing a mechanistic framework to explain clinically observed sex differences in plaque biology and to guide targeted functional interrogation.

Anthracycline chemotherapeutics increase risk for cardiovascular disease. The early molecular events that link inherited risk to cardiac dysfunction remain poorly defined. We therefore exposed iPSC-derived cardiomyocytes from six individuals to three anthracyclines and an anthracenedione, which act as topoisomerase inhibitors (TOP2i) to induce DNA damage, and generated proteomics data following three and 24 hours of TOP2i treatment. We constructed a 19 module co-expression network and identified four protein modules that associate with response to all TOP2i. Integration with a co-expression network generated from paired transcriptomic data revealed that the two most drug-responsive protein modules are preserved in the RNA network. The most preserved RNA module is enriched for p53 motifs in associated active regulatory regions and p53 target genes, which propagates to enrichment of p53 targets in the most drug-responsive preserved protein module. Integration of the protein network with genome-wide association studies across hundreds of cardiovascular traits identified a preserved protein module enriched for genetic risk of atrial fibrillation, PR interval, and longitudinal strain, suggesting that the molecular response to TOP2i is linked to genetic variation influencing cardiac electrophysiology and contractile performance. Differential protein abundance in this module associates with impaired calcium handling in cardiomyocytes thereby linking molecular effects to cellular decline. Together, these results define a TOP2i-induced gene regulatory response propagated to the proteome that underlies early cardiotoxicity, and demonstrate how a multi-level network architecture can provide insight into genetically mediated susceptibility to drug-induced stress.

Pulmonary valve stenosis (PVS) is the most common congenital heart defect in Noonan syndrome (NS) and related RASopathies, yet the molecular mechanisms linking pathogenic variants to the valve pathology remain poorly defined. Here, we utilized a human iPSC-based valve differentiation platform to generate the cardiac valve cell lineages--including fibrosa and spongiosa valve interstitial cell (VIC) subtypes. CRISPR-edited iPSCs harboring NS gain-of-function RAS/MAPK and Noonan syndrome with multiple lentigines (NSML) dominant-negative RAS/MAPK variants exhibited early defects in mesodermal and endocardial specification in all genotypes. Additionally, NS-iPSC endocardial cells exhibited defects in endothelial-to-mesenchymal transition (EndMT) specifically towards fibrosa VICs, which was most pronounced in PTPN11N308D (N308D) cells. Single-cell transcriptomics revealed widespread dysregulation of extracellular matrix (ECM) programs in N308D fibrosa VICs, including increased expression of collagens and proteoglycans, as well as dysregulation of multiple genes involved in ECM remodeling. We also detected activation of RAS-MAPK, TGF{beta}, and fibrosis-associated pathways in our transcriptional dataset. Mass spectrometry-based phosphoproteomics confirmed coordinated increases in ERK, PKC, and stress-related kinases, as well as enhanced activity of the TGF{beta} receptor. Functionally, N308D fibrosa VICs exhibited exaggerated upregulation of ECM genes in the presence of TGF{beta}2 ligand, suggesting that these cells are hypersensitive to TGF{beta} stimulation. Furthermore, we demonstrated that this pathological ECM-program occurs independently of BAMBI, a negative regulator of TGF{beta} signaling that was found to be decreased in N308D fibrosa VICs. Lastly, we performed histopathological analyses of stenotic pulmonary valves from two NS infants, which demonstrated marked overproduction and disorganization of ECM, mirroring the findings from our iPSC-based disease model. Together, our data reveal a central mechanism where NS-associated alleles sensitize fibrosa VICs to TGF{beta}, which leads to aberrant downstream signaling and drives the pathological ECM program in NS-associated PVS.

Atherosclerotic plaques accumulate low-density lipoprotein (LDL) together with antibodies targeting LDL and its apolipoprotein B (apoB) component. Given the association between IgG and plaque vulnerability, we hypothesized that apoB-specific immune complexes actively promote plaque destabilization. Using immunohistochemistry in carotid endarterectomy specimens, we quantified antibody deposition across morphologically defined plaque regions, and measured apoB reactivity and immune complex levels in matched plaque and plasma samples. IgG deposition was strongly associated with thin fibrous caps, reduced collagen content, and higher overall plaque vulnerability. Symptomatic patients exhibited increased apoB-specific IgG and reduced apoB-IgG immune complexes within plaques, indicating enhanced IgG recycling and heightened inflammatory activity. The neonatal Fc receptor (FcRn) was predominantly expressed by CD163+ macrophages, and mediated antibody recycling, LDL uptake, and production of tumor necrosis factor (TNF) and matrix metalloproteinase-9 (MMP-9) in vitro. Plaque FcRn expression increased with age and correlated with mediators of vulnerability, including collagen-degrading enzymes and pro-inflammatory cytokines. Ex vivo treatment of human plaques with a clinically used FcRn-blocking monoclonal antibody reduced IgG recycling and suppressed TNF and MMP-9 production. These findings identify FcRn-dependent antibody recycling as a contributor to inflammatory plaque vulnerability and highlight FcRn as a potential therapeutic target in atherosclerosis.

Heart failure with preserved ejection fraction (HFpEF) is an increasingly common cause of morbidity and mortality in older adults that is driven by cardiac and non-cardiac mechanisms. Physical rehabilitation improves frailty and functional capacity in HFpEF, though underlying mechanisms remain less clear. We quantified >5,000 circulating proteins across two randomized clinical trials of rehabilitation in HFpEF (REHAB-HF, SECRET-II), identifying proteins associated with prognostic measures of physical function (short physical performance battery, 6-minute walk distance) and protein changes after rehabilitation. Using an artificial intelligence (AI)-enabled multiplex network analysis (MENTOR-IA), we identified biologically plausible networks central to this "physical function proteome," including endothelial remodeling, mitochondrial metabolism, calcium handling, and immune modulation. Expression of prioritized proteins at the transcriptional level localized to heart, skeletal muscle, and brain tissue, with several cognate transcripts implicated in frailty via tissue-specific transcriptome-wide genetic association studies. In addition, using novel human genetic approaches, we implicated select proteins as mediating tissue-specific genetic effects on frailty. These findings motivated us to construct multi-protein signatures of physical function, which correlated with functional changes observed with rehabilitation in REHAB-HF and SECRET-II and that were associated with heart failure and multi-dimensional clinical outcomes in >26,000 individuals. These findings collectively delineate a multi-system molecular program underlying physical function impairment and rehabilitation response in HFpEF, offering insights into potential precision risk estimators and therapeutic targets for surveillance and promotion of physiologic resilience.

Myocardial infarction and heart failure are leading global causes of mortality. Chronic {beta}-adrenergic receptor ({beta}ARs) activation in cardiomyocytes promotes heart failure via Gs signaling after myocardial infarction, whereas {beta}2AR activation may also provide cardiac protection and repair through alternative pathways. Macrophages play a pivotal role in cardiac repair, and {beta}2AR has been reported to signal via the hematopoietic-specific G15 in these cells. We aim to characterize signaling bias between Gs and G15 downstream of {beta}2AR and to elucidate their roles in macrophage polarization. Using TRUPATH BRET assays, we demonstrate that several {beta}2AR agonists activate G15 with at least an order of magnitude greater potency than Gs. Clinically used {beta}-blockers exhibit differential inhibition on these two pathways. Transcriptome analysis of THP-1-derived macrophage-like cells treated with the {beta}2AR agonist clenbuterol revealed a mixed transcriptional profile with enrichment of both M1 inflammatory and M2/repair-associated gene sets. Knockdown experiments showed that Gs suppresses M1-like phenotypes while enhancing M2-like phenotypes, whereas G15 is specifically required for M2-like regulation. Pharmacological blockade of the Gs-adenylyl cyclase interaction produced opposing effects on M1/M2 signatures compared to Gs knockdown, while producing concordant effects on the repair-associated gene sets. These findings characterize the distinct pharmacological profiles of {beta}2AR ligands toward Gs and G15 and reveal how {beta}2AR agonism modulates macrophage function through dual-transducer signaling. Significance statement{beta}2AR displays marked signaling bias toward the hematopoietic-specific G15 over canonical Gs, with multiple clinically relevant agonists activating G15 at [&ge;]10-fold higher potency. Clinically used {beta}-blockers exhibit differential inhibition--timolol preferentially blocks Gs, while labetalol has reduced efficacy against G15--while in macrophages, Gs suppresses pro-inflammatory M1 programs and supports M2 repair signatures, whereas G15 specifically reinforces M2-associated and tissue-repair transcriptional modules, revealing a dual-transducer mechanism that may enhance macrophage-mediated cardiac repair after myocardial infarction and support biased {beta}2AR ligands.

Heart attacks and strokes are late-stage complications of rupture of unstable atherosclerotic plaques. Stable plaques contain stabilizing matrix-producing fibrotic cells, largely smooth muscle cell (SMC)-derived. The molecular drivers of SMC phenotypic transitions to beneficial fibrotic or destabilizing inflammatory and calcifying phenotypes are unclear. Since atherosclerosis develops over decades, there is extensive interest in identifying dietary alterations that enhance plaque stability. We demonstrate that SMC acquire a fibrotic phenotype dependent on glutamine-derived metabolites supporting both catabolism and collagen synthesis. Moreover, dietary glutamine restriction decreases mortality of mice susceptible to atherosclerotic plaque rupture. Lesions from glutamine-restricted mice are smaller and have increased SMC investment. This study identifies dietary glutamine as a driver of cardiovascular mortality, suggesting a new strategy for reducing late-stage complications of atherosclerosis.

Cardiovascular diseases are a major global health burden, demanding phenotyping frame-works that can match the scale and complexity of contemporary mouse genetics. Here, we introduce EchoVisuALL, an AI-enabled pipeline for automated high-throughput transthoracic echocardiography (TTE) coupling deep-learning-based left-ventricular segmentation with data reporting. Across 65,000 recordings from over 18,000 mice, including single-gene knockouts from the International Mouse Phenotyping Consortium, the framework quantified cardiac morphology and function with minimal operator dependency and high reliability, validated against an expert-curated gold standard dataset. By extracting quantitative parameters across the cardiac cycle, EchoVisuALL in combination with multi-dimensional clustering uncovered nonlinear phenotypic relationships and revealed 37 of 715 genes associated with significant cardiac abnormalities, encompassing well-known human disease genes as well as 12 previously unrecognized candidates, including Cep70, Acot12, Atp8b3, Eea1, Kctd2, and Tspan15. These genotype-phenotype associations are involved in myocardial energetics, membrane biology, and cardiac remodeling. We demonstrate the potential of EchoVisuALL to move beyond image segmentation by delivering a standardized, quantitative foundation for scalable downstream analyses, enabling the discovery of novel cardiac disease genes.

Myocardial infarction (MI) triggers a systemic neutrophil response, yet the roles of distinct neutrophil subsets in cardiac remodeling remain unclear. Studying this requires murine models that accurately mirror human neutrophil dynamics. Here, we show that a minimally invasive intact-chest MI model is more pathophysiologically relevant than the standard open-chest approach for investigating post-MI immune responses. In the open-chest model, surgical trauma disrupts bone marrow homeostasis, releases large numbers of immature neutrophils, and masks MI-specific immune mechanisms. In contrast, the intact-chest model preserves bone marrow integrity and induces only a modest rise in circulating immature neutrophils, closely reflecting MI patient profiles. We further demonstrate that accumulation of immature neutrophils in the infarcted heart exacerbates cardiac dysfunction. Beyond neutrophils, the overall cardiac immune landscape differs markedly between both models. Collectively, our findings establish the intact-chest model as superior for studying post-MI inflammation and reveal immature neutrophils as mediators of adverse cardiac remodeling.

While dysbiosis and inflammation were previously implicated in cardiovascular diseases, the circuits of how microbiota drives distant perivascular innervation, neuroinflammation and atherosclerosis remains unknown. Here, we report that IL-17RC signaling in intestine protects from atherosclerosis controlling intestinal barrier and microbiota, and loss of IL-17RC in intestinal epithelial cells alters microbiota, enhances perivascular innervation and aortic inflammation, augmenting the disease. Neuronal outgrowth is functionally dependent on microbiota and is essential for neuroinflammation and augmentation of atherosclerosis as chemical denervation reduces inflammation, macrophage activation and disease progression. Microbiota-dependent IL-17A producing {gamma}{delta} T cells accumulate in aorta to promote neuronal outgrowth and activation that can be reversed by {gamma}{delta} T cell blockade. Perivascular neuron activation is further dependent on cell autonomous IL-17 signaling as IL-17RC ablation in sympathetic neurons protected mice from microbiota-driven atherosclerosis. Together, our data illuminate how intestinal cytokine signaling distantly restrains neuroimmune interactions in aorta and uncovers a novel link between IL-17 signaling, microbiota, perivascular innervation and neuroimmune pro-inflammatory crosstalk instrumental for atherosclerosis progression. SummaryIL-17RC signaling regulates intestinal dysbiosis and perivascular neuronal outgrowth that modulates inflammation in atherosclerosis.