BACKGROUND:Cardiac hypertrophy is a major contributor to heart failure development, making its prevention and treatment critical for reducing heart failure-associated mortality. Although alternative splicing is recognized as a key regulatory mechanism in myocardial hypertrophy, the precise pathways involved remain incompletely defined. METHODS:To investigate the role of MBNL2 (muscleblind-like protein 2) in the heart, we overexpressed MBNL2 in cardiomyocytes via adeno-associated virus serotype 9 delivery. In addition, cardiomyocyte-specific MBNL2 knockout mice were generated, and transverse aortic constriction surgery or isoproterenol injection was performed to induce cardiac hypertrophy and dysfunction in mice. The underlying mechanisms were further investigated using RNA sequencing, alternative splicing analysis, and RNA immunoprecipitation. RESULTS:MBNL2 expression was significantly increased in the heart tissues from patients with ischemic cardiomyopathy and in mice with cardiac hypertrophy. Cardiac-specific overexpression of MBNL2 induced cardiac hypertrophy and dysfunction. Mechanistically, MBNL2 promoted exon 9 skipping of TPM3 (tropomyosin 3), generating the TPM3 isoform lacking exon 9 (TPM3-Δe9). In neonatal mouse cardiomyocytes, TPM3-Δe9 knockdown partially reduced oxidative stress and mitigated mitochondrial damage induced by MBNL2 overexpression. Adeno-associated virus serotype 9-mediated knockdown of TPM3-Δe9 partially attenuated cardiac hypertrophy in MBNL2-overexpressing mice. Notably, cardiac-specific MBNL2 knockout or TPM3-Δe9 knockdown in mice attenuated cardiac dysfunction induced by transverse aortic constriction. Further, we found that elevated TPM3-Δe9 was associated with RNF20 (ring finger protein 20) and was accompanied by reduced RNF20 interaction with NCoR1 (nuclear receptor corepressor 1), increased NCoR1 expression, and decreased PPARα (peroxisome proliferator-activated receptor alpha) signaling. The protective effects of MBNL2 or TPM3-Δe9 knockdown in hypertrophic cardiomyocytes were partially reversed by treatment with the PPARα inhibitor GW6471. CONCLUSIONS:This study uncovers a novel, critical role for MBNL2 in pathological cardiac hypertrophy through regulation of TPM3 alternative splicing and mitochondrial function, highlighting MBNL2 as a potential therapeutic target for cardiac hypertrophy and dysfunction.
BACKGROUND:Patients with inflammatory bowel disease are at increased risk of cardiovascular disease, yet the mechanisms linking chronic intestinal inflammation to cardiac dysfunction remain poorly understood. Inflammatory bowel disease is characterized by profound gut microbiota dysbiosis, which we hypothesize drives systemic immune dysregulation and contributes to cardiac dysfunction. METHODS:A chronic colitis mouse model was used to assess gut microbiota dysbiosis, systemic immune cell metabolism, and cardiac remodeling. Cardiac outcomes were evaluated by echocardiography, histology, and molecular analyses. Mechanisms were examined using fecal microbiota transplantation, immune cell depletion, exosome transfer, bone marrow chimeras, RNA sequencing, coimmunoprecipitation, confocal microscopy, and siRNA-mediated gene silencing. RESULTS:Chronic dextran sulfate sodium colitis induced cardiac dysfunction, hypertrophy, and fibrosis in mice. These changes were accompanied by sustained gut microbiota dysbiosis, metabolic reprogramming, and mitochondrial dysfunction in circulating immune cells. Fecal microbiota transfer experiments demonstrated that colitis-associated microbiota were sufficient to reprogram systemic immune cells and promote cardiac dysfunction. Immune cell depletion studies identified macrophages as key mediators of colitis-associated cardiac injury. Colitis increased systemic lipopolysaccharide translocation; bone marrow chimera experiments demonstrated that hematopoietic TLR4 (toll-like receptor 4) signaling was required for immune cell metabolic remodeling and cardiac dysfunction during chronic colitis. Transcriptomic analysis identified GBP2b (guanylate-binding protein 2b/GBP1, hereafter referred to as GBP1) as a key downstream effector of lipopolysaccharide TLR4 signaling. Upon lipopolysaccharide stimulation, GBP1 localized to mitochondria, where it interacted with DRP1 (dynamin-related protein 1) and FIS1 (fission 1 protein) to promote mitochondrial fission, oxidative stress, and enhanced immune cell migration into the heart. In addition, GBP1 was secreted via exosomes, which were taken up by cardiomyocytes and contributed to hypertrophic remodeling and cardiac dysfunction. CONCLUSIONS:These findings establish the lipopolysaccharide TLR4-GBP1 axis as a key driver of colitis-associated cardiovascular dysfunction and highlight this pathway as a promising therapeutic target for reducing cardiovascular risk in patients with inflammatory bowel disease.
BACKGROUND:Elevated 1-hour glucose levels during an oral glucose tolerance test strongly predict type 2 diabetes (T2D) and cardiovascular disease. We investigated whether the fasting blood metabolome predicting 1-hour glucose could be a target for improving β-cell function, long-term glycemic trajectories, and reducing the risks of T2D and coronary heart disease. We also investigated whether plasma microRNAs derived from key metabolic organs regulate changes in a metabolomic risk score (MRS) for predicting 1-hour glucose. METHODS:Untargeted blood metabolomics and a frequently sampled 75-g oral glucose tolerance test were performed in participants from the OmniCarb trial (n=162). In an independent weight-loss dietary intervention trial (POUNDS Lost [Preventing Overweight Using Novel Dietary Strategies]), temporal changes in MRS and plasma microRNAs measured by genome-wide sequencing were analyzed. In addition, associations of MRS at baseline and its 10-year changes with long-term risk of incident T2D and coronary heart disease were prospectively investigated in the NHS (Nurses' Health Study). RESULTS:We created a fasting blood MRS for predicting 1-hour glucose (Pearson r=0.8) and found significant associations with half-day (diurnal) postprandial glucose excursions and insulin secretion after 5-week controlled feeding interventions varying in carbohydrate amount and glycemic index. In the POUNDS Lost trial, diet-induced changes in MRSs were related to 2-year trajectories of glucose metabolism; circulating microRNAs regulating cardiometabolic abnormalities were pivotal factors influencing these changes. In the NHS, women in the top 20% of MRS had a multivariate-adjusted relative risk of 3.80 (95% CI, 2.22-6.51) for T2D and 1.48 (95% CI, 1.04-2.12) for coronary heart disease compared with those in the lowest 20%. In addition, 10-year increases in plasma metabolites related to 1-hour glucose were linearly associated with a higher risk of T2D. CONCLUSIONS:Our findings indicate that fasting blood metabolomic signatures predicting elevated 1-hour glucose reflect disease pathophysiology and could be targets for preventing T2D and coronary heart disease.
BACKGROUND: Mutations in DSP , which encodes the protein desmoplakin, lead to cardiomyopathy with unusually high penetrance that presents with arrhythmias, fibro-fatty infiltration, and eventually heart failure. However, the precise mechanism of contractile dysfunction and dilation is incompletely understood. Here, we investigate the pathogenesis of DSP -R451G, a missense mutation that results in complete degradation of desmoplakin protein. METHODS: We use 3 complementary models to characterize desmoplakin-linked cardiomyopathy: induced pluripotent stem cell-derived engineered heart tissue expressing R451G desmoplakin, a heterozygous Dsp WT/R451G knock-in mouse, and left-ventricular biopsy specimens. Tissue-engineered constructs are used to characterize contractility, calcium handling, sarcomere length, and cell signaling. These results are corroborated in the R451G mouse. To expand the generalizability of the findings, we compare them to those from human heart biopsies bearing 3 different desmoplakin mutations. RESULTS: Using induced pluripotent stem cell-derived engineered heart tissue and isolated mouse ventricular cardiomyocytes, we recapitulate a disease phenotype consistent with desmoplakin cardiomyopathy and identify shortened resting sarcomere length as a pathogenic mechanism for contractile dysfunction. Phosphorylation of Src and protein kinase C underlies sarcomere shortening in mutant tissues, and pharmacological inhibition of these kinases rescues sarcomere length. Notably, these sarcomeric and biochemical hallmarks are also present in human hearts bearing 3 different desmoplakin mutations. We next identify redistribution of mechanical force at cardiomyocyte junctions as a proximal factor that may promote mechanoactivation of proto-oncogene tyrosine-protein kinase Src. Finally, we rescue sarcomere length and contractile function in DSP -mutant engineered heart tissue with dasatinib, a Food and Drug Administration-approved receptor tyrosine kinase inhibitor. CONCLUSIONS: Our study reveals a mechanism by which a desmosomal mutation affects cardiomyocyte function at the sarcomere level through activation of key signaling pathways that have not previously been implicated in desmoplakin cardiomyopathy.
BACKGROUND:Translational control of gene expression is crucial in cardiomyocytes, particularly in response to hypertrophic stimuli. The ERK (extracellular signal-regulated kinase) pathway plays a key role in inducing cardiac hypertrophy and regulating specific protein translation. However, it remains unclear how this specificity is achieved, and the spatiotemporal regulation of protein translation is not fully understood. METHODS:We used SINAP (single-molecule imaging of nascent peptide) reporters to visualize and analyze the translation dynamics in single adult rat ventricular cardiomyocytes and tracked active translation sites at high spatiotemporal resolution. We also examined the effects of adrenergic stimulation and the role of the ERK pathway in translation localization. RESULTS:Our findings revealed that translation sites are primarily localized near Z-lines in cardiomyocytes, with some sites being highly dynamic and moving during translation. The 3' untranslated regions did not significantly change the localization of translation. Many translation sites colocalized with microtubules, and their movement predominantly occurred along microtubular tracks. Adrenergic stimulation led to a transient shift in translation activity toward the perinuclear region, peaking at 12 hours and requiring ERK pathway activity for this localization change. This shift is part of the hypertrophic response and is required for early translation of genes such as Nppa. CONCLUSIONS:Our high-resolution single-cell study demonstrates that protein translation in cardiomyocytes is dynamic and responsive to hypertrophic stimuli in an ERK-dependent manner. The localized translation mechanism allows cardiomyocytes to rapidly adapt to changing environments by preferentially translating mRNAs in the perinuclear region. These findings provide new insights into the spatial regulation of translation in cardiomyocytes and its role in cardiac hypertrophy.
BACKGROUND:Peripheral artery occlusive disease (PAOD) is characterized by limb ischemia, heightened inflammation, and a substantial risk of mortality and amputation. Coordinated regulation of inflammation resolution and angiogenesis represents a promising therapeutic strategy. Given the established roles of FPRs (formyl peptide receptors) in inflammation resolution, we investigated their contribution to the integrated control of inflammation and angiogenesis in PAOD. METHODS:Using a murine hindlimb ischemia model and clinical blood samples, we identified FPR1 as a key regulator of PAOD. We evaluated hindlimb perfusion recovery and inflammatory responses in Fpr1-deficient mice. The therapeutic potential of an FPR1 agonist was assessed, and RNA sequencing was used to elucidate underlying mechanisms. FPR1‑dependent angiogenic responses were further confirmed in vivo using neutrophil‑specific Fpr1-knockout mice and in vitro using neutrophils isolated from Fpr1-deficient mice and individuals carrying the FPR1 rs867228 variant. RESULTS:We identified FPR1 as a pivotal regulator in PAOD. Fpr1 deficiency led to exaggerated inflammation and impaired angiogenesis, whereas FPR1 activation by a biased agonist, Cmpd17b, attenuated inflammation and improved perfusion recovery through a neutrophil‑dependent mechanism. Mechanistically, the Cmpd17b shifted neutrophil signaling away from Ca2+‑dependent proinflammatory pathways toward IL‑10 (interleukin-10) and VEGF-A (vascular endothelial growth factor‑A) production. Further analysis revealed that FPR1 activation in neutrophils promotes CCL2 (C-C motif chemokine ligand 2) release, which may upregulate HMOX1 (heme oxygenase 1) in endothelial cells to enhance angiogenesis. We also demonstrated that FPR1 rs867228 confers protection in human PAOD by favoring pro-resolving responses and enhancing CCL2-mediated angiogenesis. CONCLUSIONS:FPR1 is a central regulator of inflammation and angiogenesis in PAOD. Biased FPR1 activation engages a neutrophil/CCL2-endothelial/HMOX1 axis to resolve inflammation and promote angiogenesis, supporting its therapeutic potential in PAOD.
BACKGROUND: Salt-sensitivity of blood pressure is an independent risk factor for cardiovascular diseases, yet the molecular pathways linking dietary sodium to immune activation and hypertension remain poorly defined. We previously demonstrated that sodium entry into antigen-presenting cells via the ENaC (epithelial sodium channel) promotes inflammation and salt-sensitivity of blood pressure. AP-1 (activator protein-1; c-FOS, FOSB, c-JUN, JUNB, JUND) regulates inflammatory signaling, but its role in salt-sensitivity of blood pressure has not been elucidated. We hypothesized that high salt drives AP-1-mediated inflammatory activation in antigen-presenting cells, contributing to immune dysfunction and hypertension. METHODS: Using SV129 salt-sensitive mice, we assessed blood pressure responses and profiled immune cell phenotypes under normal- and high-salt conditions by flow cytometry. RNA-seq was performed on human monocytes exposed to high salt in vitro. In a clinical study, we enrolled prehypertensive subjects and performed an inpatient salt-loading/depletion protocol to characterize AP-1 gene expression signatures in salt-sensitive versus salt-resistant individuals. To test causality, we adoptively transferred PBMCs from salt-sensitive, salt-resistant, and salt-sensitive individuals pretreated with T5224 (a selective AP-1 inhibitor) into immunodeficient NSG-( K b D b ) null ( IA ) null humanized mice, followed by assessment of blood pressure, vascular reactivity, kidney function, and immune infiltration. RESULTS: High salt robustly induced AP-1 gene expression in murine monocytes. In humans, salt-sensitive but not salt-resistant subjects exhibited concordant increases in AP-1 gene expression and blood pressure during salt loading. PBMCs from salt-sensitive individuals promoted greater tissue infiltration, AP-1 activation, and immune-mediated renal and vascular dysfunction in humanized mice compared with PBMCs from salt-resistant individuals. Strikingly, pretreatment of salt-sensitive PBMCs with T5224 abrogated these effects, preserving normal renal and vascular function despite high-salt exposure. CONCLUSIONS: These findings identify AP-1 as a key transcriptional driver linking dietary sodium, immune activation, and salt-sensitivity of blood pressure. Targeting AP-1 signaling mitigates immune-mediated renal and vascular injury, highlighting a novel mechanistic pathway and a therapeutic target for salt-sensitive hypertension.
Platelets are central to hemostasis and thrombosis. Excessive platelet activation contributes to arterial thrombotic events, including myocardial infarction, ischemic stroke, and complications of peripheral artery disease, whereas excessive platelet inhibition increases bleeding risk. Antiplatelet therapy remains a cornerstone of secondary prevention in atherosclerotic and thrombotic cardiovascular diseases, yet current treatment paradigms do not fully account for biological heterogeneity in platelet function, including differences related to sex, age, hormonal status, and disease context. This state-of-the-art review examines current antiplatelet strategies, fundamental mechanisms of signal transduction, clinical indications, and limitations of preclinical and clinical studies, with a focus on sex-specific considerations and opportunities for personalized antiplatelet therapy. Aspirin and P2Y12 receptor antagonists are widely used for secondary prevention. However, women have been underrepresented in many pivotal clinical trials, limiting the precision of sex-specific estimates of efficacy and bleeding risk. In parallel, commonly used preclinical models often fail to recapitulate the physiological conditions in which platelets interact with the vasculature, leukocytes, and soluble factors. Emerging therapeutic approaches seek to refine platelet inhibition by targeting pathways that reduce thrombotic risk while preserving hemostasis. Advancing antiplatelet therapy will require integration of mechanistic platelet biology with diverse clinical trial populations, standardized platelet phenotyping, and disease-specific approaches that account for how platelet function is altered across health and vascular disease.
Cardiovascular diseases remain the leading global cause of mortality, highlighting the need for improved early detection and targeted interventions. Extracellular vesicles (EVs) are nano-sized, bilipid-layered particles released by all cell types that carry RNAs, proteins, lipids, and metabolites reflective of their parent cells. They mediate intercellular communication by transferring cargo that alters recipient cell transcriptomic and proteomic states, and this property may be leveraged for therapeutic delivery. This review provides a comprehensive, cardiovascular disease-focused synthesis of EV biology with emphasis on what is clinically actionable and mechanistically novel. The review describes EV biogenesis and their multiomic cargo composition, followed by tissue-resolved and cell type-resolved EV signaling across cell types relevant to cardiovascular disease. A dedicated section addresses EV-mediated interorgan crosstalk across the heart-kidney, heart-liver, brain-heart, and adipose-heart axes as a systems-level framework for cardiometabolic disease. We next turn to translational applications, describing EV cargo composition under pathological conditions with implications for disease-related signaling and the potential for biomarker development. For liquid biopsy applications, the review introduces a 3-tier evidence framework classifying circulating EV biomarkers by clinical validation status, supported by a practical preanalytical checklist for cardiovascular plasma studies. Engineered, stem cell-derived, and RNA-loaded EV therapeutic modalities are evaluated, and active clinical trials are catalogued along with key challenges in cargo loading, biodistribution, immunogenicity, and regulatory standardization. We conclude with a structured future directions and perspectives section identifying the most tractable open questions required to advance EVs from discovery to cardiovascular clinical practice.
Mitochondrial heteroplasmy represents a fundamental determinant of mitochondrial function and disease, yet its consequences vary across different tissues. Although mitotic tissues possess mechanisms, such as cell division and mitochondrial turnover, to dilute or remove deleterious variants, postmitotic tissues lack this renewal capacity and are disproportionately vulnerable. Neuromuscular and neurodegenerative disorders have illustrated the impact of heteroplasmic mutations, but the (postmitotic) heart remains underexplored. Current reliance on blood-derived samples provides only an indirect view of cardiac heteroplasmy, highlighting the need for alternative approaches, such as endomyocardial biopsies and human induced pluripotent stem cell-derived cardiomyocytes. Expanding cardiac-focused research is essential for identification, clarifying pathogenesis, improving risk stratification, and guiding patient monitoring. Emerging therapies, including mitochondrial transplantation and mitochondrial-targeted DNA editing, demonstrate potential to modulate heteroplasmy and restore equilibrium. Integrating these strategies with precision medicine will be vital for addressing tissue-specific vulnerabilities. Ultimately, bridging the gap in cardiac heteroplasmy research will be critical for translating basic mitochondrial biology into meaningful clinical advances.
BACKGROUND: Anthracycline-induced cardiotoxicity (AIC) limits life-saving chemotherapy and is driven by early metabolic remodeling. The nuclear receptor ERRα (estrogen-related receptor α) is a master regulator of cardiac energy metabolism, but the temporal dynamics of its downregulation, its causal role in AIC pathogenesis, and whether it can be pharmacologically activated to confer protection remain undefined. METHODS: We performed temporal protein analysis in a porcine AIC model. Using cardiomyocyte-specificgain- and loss-of-function mouse models, we assessed the causal role of ERRα. Mechanistic studies included ChIP-qPCR, reporter assays, and microscale thermophoresis to investigate the natural compound formononetin. Human breast cancer patient-derived organoids were used to evaluate anticancer activity. RESULTS: ERRα expression was selectively downregulated in AIC pig hearts and cardiac tissue from chemotherapy-treated patients. Temporal analysis in pigs revealed that ERRα reduction occurred at the subclinical (6-week) stage, preceding overt cardiac dysfunction. Cardiomyocyte-specific ERRα overexpression activated mitochondrial gene programs, enhanced fatty acid oxidation, and preserved systolic function after doxorubicin challenge, whereas ERRα knockdown exacerbated bioenergetic failure and cardiac dysfunction. Through drug screening, we identified formononetin as a potent and selective ERRα agonist. Formononetin enhanced ERRα transcriptional activity, improved mitochondrial metabolism, and protected against AIC in both murine and porcine models. Mechanistically, ChIP-qPCR demonstrated increased ERRα occupancy at target gene promoters, and microscale thermophoresis confirmed direct binding of formononetin to the ERRα/PGC-1α complex, indicating allosteric stabilization. Finally, in human breast cancer patient-derived organoids, formononetin alone reduced viability and proliferation, and combined with doxorubicin further enhanced antitumor efficacy. CONCLUSIONS: ERRα downregulation is a causal early event in the pathogenesis of AIC. Formononetin acts as a first-in-class selective ERRα activator that improves cardiac metabolism and function while retaining anticancer activity, supporting its potential as a dual-action cardioprotective agent during anthracycline therapy.
BACKGROUND:Recent studies have revealed heterogeneity among ribosomes. Pathological cardiac hypertrophy is characterized by profound alterations in translation. However, how ribosome heterogeneity contributes to this process remains largely unclear. METHODS:We used translating ribosome affinity purification coupled with mass spectrometry to profile ribosome-interacting proteins. Cardiomyocyte-specific gene manipulation was achieved through either genetic knockout or adeno-associated virus-mediated overexpression. Pathological cardiac hypertrophy was induced by transverse aortic constriction surgery in vivo and by phenylephrine stimulation in vitro. RESULTS:The cardiomyocyte-specific ribosome proteomics indicated dynamic alterations in ribosome-interacting proteins during pathological hypertrophy. Notably, multiple proteins associated with ribosome stalling were detected in the ribosome-interactome of hypertrophic hearts. Among these, we verified that CDK5RAP3 (CDK5 regulatory subunit-associated protein 3) exhibited the most specific ribosome binding in hypertrophic hearts. CDK5RAP3 was upregulated and recruited to ribosomes during pathological hypertrophy. It promoted RPL26 (ribosomal protein L26) UFMylation and ribosome-associated quality control on the mitochondrial surface. In vitro, CDK5RAP3 knockdown exacerbated cardiomyocyte hypertrophy induced by phenylephrine, whereas its overexpression attenuated it. In vivo, cardiomyocyte-specific CDK5RAP3 knockout promoted, while adeno-associated virus-mediated overexpression suppressed pathological cardiac hypertrophy induced by transverse aortic constriction. Mechanistically, ribosome stalling on the mitochondrial surface was exacerbated in hypertrophic hearts of both humans and mice, which was associated with impaired mitochondrial protein import. CDK5RAP3 enhanced ribosome-associated quality control, alleviated ribosome stalling, and restored mitochondrial protein import, thereby improving mitochondrial function. Notably, mitochondrial import of PDP1 was maintained by CDK5RAP3-mediated ribosome-associated quality control. Knockdown of PDK (pyruvate dehydrogenase kinase) 1/2, functional antagonists of PDP1, reversed cardiomyocyte hypertrophy caused by CDK5RAP3 deficiency. CONCLUSIONS:This study identifies CDK5RAP3-mediated ribosome-associated quality control on the mitochondrial surface as a critical protective mechanism that preserves protein import and mitochondrial function during pathological cardiac hypertrophy.
BACKGROUND:Pathogenic immune-cardiac crosstalk underlies maladaptive remodeling in chronic heart failure, yet therapies directly targeting this axis are lacking. Glycoconjugates, which are crucial for signal transduction and extracellular matrix integrity, represent an underexploited therapeutic avenue. This study sought to define the role of glycoconjugate-metabolizing enzymes at the immune-cardiac interface and evaluate their translational potential. METHODS:We performed integrative analyses of bulk and single-cell RNA sequencing data from failing human and mouse hearts. Employing mouse models of pressure overload (transverse aortic constriction) and ischemia-reperfusion, we used global and mast cell (MC)-specific gene deletion, bone-marrow chimeras, and pharmacological neutralization. Mechanistic insights were gained through multiomics profiling, including RNA-seq, ATAC-seq, CUT&Tag, and proteomics. RESULTS:The ganglioside GD3 synthase, St8sia1, was selectively induced in cardiac MCs during pathological remodeling in both mice and humans. MC-specific or hematopoietic deletion of St8sia1 preserved ventricular function, attenuated fibrosis, and markedly reduced neutrophil and Ly6C+ monocyte recruitment after transverse aortic constriction and ischemia-reperfusion. Therapeutic neutralization of GD3 with the clinical-grade monoclonal antibody R24 improved cardiac function and diminished scar formation after ischemia-reperfusion. Mechanistically, GD3 bound specific histone variants, such as H2A.Z and H3.3C, thereby reprogramming chromatin accessibility to activate proinflammatory and profibrotic transcriptional programs in MCs. Consequently, GD3 inhibition suppressed MC degranulation, disrupted pathogenic MC-cardiomyocyte/fibroblast crosstalk, and preserved reparative macrophage populations. CONCLUSIONS:The MC-restricted St8sia1-GD3 axis functions as a glyco-epigenetic checkpoint driving maladaptive cardiac remodeling. Targeting this axis represents a translatable immunomodulatory strategy to prevent the progression to chronic heart failure.
