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BACKGROUND: The metabolic environment plays a crucial role in the development of heart failure (HF). Our prior research demonstrated that myo-inositol, a metabolite transported by the sodium-myo-inositol co-transporter 1 (SMIT-1), can induce oxidative stress and may be detrimental to heart function. However, plasmatic myo-inositol concentration has not been comprehensively assessed in large cohorts of patients with heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF). METHODS: Plasmatic myo-inositol levels were measured using mass spectrometry and correlated with clinical characteristics in no HF subjects and patients with HFrEF and HFpEF from Belgian (male, no HF, 53%; HFrEF, 84% and HFpEF, 40%) and Canadian cohorts (male, no HF, 51%; HFrEF, 92% and HFpEF, 62%). FINDINGS: Myo-inositol levels were significantly elevated in patients with HF, with a more pronounced increase observed in the HFpEF population of both cohorts. After adjusting for age, sex, body mass index, hypertension, diabetes, and atrial fibrillation, we observed that both HFpEF status and impaired kidney function were associated with elevated plasma myo-inositol. Unlike HFrEF, abnormally high myo-inositol (≥69.8 µM) was linked to unfavourable clinical outcomes (hazard ratio, 1.62; 95% confidence interval, [1.05-2.5]) in patients with HFpEF. These elevated levels were correlated with NTproBNP, troponin, and cardiac fibrosis in this subset of patients. INTERPRETATION: Myo-inositol is a metabolite elevated in patients with HF and strongly correlated to kidney failure. In patients with HFpEF, high myo-inositol levels predict poor clinical outcomes and are linked to markers of cardiac adverse remodelling. This suggests that myo-inositol and its transporter SMIT1 may have a role in the pathophysiology of HFpEF. FUNDING: BECAME-HF was supported by Collaborative Bilateral Research Program Québec - Wallonie-Brussels Federation.
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In vitro testing for evaluating degradation mode and rate of candidate biodegradable metals to be used as intravascular stents is crucial before going to in vivo animal models. In this study, we show that X-ray microfocus computed tomography (microCT) presents a key added value to visualize degradation mode and to evaluate degradation rate and material surface properties in 3D and at high resolution of large regions of interest. The in vitro degradation behavior of three candidate biodegradable stent materials was evaluated: pure iron (Fe), pure zinc (Zn), and a quinary Zn alloy (ZnAgCuMnZr). These metals were compared to a reference biostable cobaltchromium (CoCr) alloy. To compare the degradation mode and degradation rate evaluated with microCT, scanning electron microscopy (SEM) and inductively-coupled plasma (ICP) were included. We confirmed that Fe degrades very slowly but with desirable uniform surface corrosion. Zn degrades faster but exhibits localized deep pitting corrosion. The Zn alloy degrades at a similar rate as the pure Zn, but more homogeneously. However, the formation of deep internal dendrites was observed. Our study provides a detailed microCT-based comparison of essential surface and corrosion properties, with a structural characterization of the corrosion behavior, of different candidate stent materials in 3D in a non-destructive way.
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Implantes Absorvíveis , Teste de Materiais , Stents , Microtomografia por Raio-X , Zinco , Zinco/química , Ligas/química , Ferro/química , CorrosãoRESUMO
PURPOSE OF REVIEW: Lipids play vital roles in platelet structure, signaling, and metabolism. In addition to capturing exogenous lipids, platelets possess the capacity for de novo lipogenesis, regulated by acetyl-coA carboxylase 1 (ACC1). This review aims to cover the critical roles of platelet de novo lipogenesis and lipidome in platelet production, function, and diseases. RECENT FINDINGS: Upon platelet activation, approximately 20% of the platelet lipidome undergoes significant modifications, primarily affecting arachidonic acid-containing species. Multiple studies emphasize the impact of de novo lipogenesis, with ACC1 as key player, on platelet functions. Mouse models suggest the importance of the AMPK-ACC1 axis in regulating platelet membrane arachidonic acid content, associated with TXA 2 secretion, and thrombus formation. In human platelets, ACC1 inhibition leads to reduced platelet reactivity. Remodeling of the platelet lipidome, alongside with de novo lipogenesis, is also crucial for platelet biogenesis. Disruptions in the platelet lipidome are observed in various pathological conditions, including cardiovascular and inflammatory diseases, with associations between these alterations and shifts in platelet reactivity highlighted. SUMMARY: The platelet lipidome, partially regulated by ACC-driven de novo lipogenesis, is indispensable for platelet production and function. It is implicated in various pathological conditions involving platelets.
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Plaquetas , Lipidômica , Lipogênese , Humanos , Plaquetas/metabolismo , Animais , Lipidômica/métodos , Acetil-CoA Carboxilase/metabolismo , Metabolismo dos Lipídeos , Ativação PlaquetáriaRESUMO
AIMS: Heart failure (HF) with preserved ejection fraction (HFpEF) is a disease associated with high morbidity and mortality, for which it is difficult to identify patients with the poorest prognosis in routine clinical practice. Carbohydrate antigen 125 (CA 125) has been shown to be a potential marker of congestion and prognosis in HF. We sought to better characterize HFpEF patients with high CA 125 levels by using a multimodal approach. METHODS AND RESULTS: We prospectively enrolled 139 HFpEF patients (78 ± 8 years; 60% females) and 25 controls matched for age and sex (77 ± 5 years; 60% females). They underwent two-dimensional echocardiography, cardiac magnetic resonance with late gadolinium enhancement [including extracellular volume (ECV) measurement], and serum measurements of CA 125 level. The primary endpoint of the study was a composite of all-cause mortality or first HF hospitalization. The prognostic impact of CA 125 was determined using Cox proportional hazard models. Median CA 125 levels were significantly higher in HFpEF patients compared with controls [CA 125: 23.5 (14.5-44.7) vs. 14.6 (10.3-21.0) U/mL, P = 0.004]. CA 125 levels were positively correlated with a congestion marker [N-terminal pro-brain natriuretic peptide (NT-proBNP) levels, Pearson's r = 0.37, P < 0.001] and markers of cardiac fibrosis estimated by both ECV (Pearson's r = 0.26, P = 0.003) and fibroblast growth factor 23 levels (Pearson's r = 0.50, P < 0.001). Over a median follow-up of 49 (22-64) months, 97 HFpEF patients reached the composite endpoint. Even after adjustment for the Meta-Analysis Global Group in Chronic risk score, a CA 125 level ≥35 U/mL was still a significant predictor of the composite endpoint [hazard ratio (HR): 1.58 (1.04-2.41), P = 0.032] and more particularly of HF hospitalization [HR: 1.81 (1.13-2.92), P = 0.014]. In contrast, NT-proBNP levels were not an independent predictor. CONCLUSIONS: CA 125 levels were significantly higher in HFpEF patients compared with controls matched for age and sex and were associated with markers of congestion and cardiac fibrosis. CA 125 levels were a strong and independent predictor of HF hospitalization in HFpEF patients. These data suggest a potential value of CA 125 as a biomarker for staging and risk prediction in HFpEF.
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Biomarcadores , Antígeno Ca-125 , Fibrose , Insuficiência Cardíaca , Imagem Cinética por Ressonância Magnética , Volume Sistólico , Humanos , Feminino , Masculino , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/diagnóstico , Antígeno Ca-125/sangue , Idoso , Prognóstico , Estudos Prospectivos , Volume Sistólico/fisiologia , Biomarcadores/sangue , Imagem Cinética por Ressonância Magnética/métodos , Fibrose/sangue , Seguimentos , EcocardiografiaRESUMO
Myocardial fibrosis is a feature found in most cardiac diseases and a key element contributing to heart failure and its progression. It has therefore become a subject of particular interest in cardiac research. Mechanisms leading to pathological cardiac remodeling and heart failure are diverse, including effects on cardiac fibroblasts, the main players in cardiac extracellular matrix synthesis, but also on cardiomyocytes, immune cells, endothelial cells, and more recently, platelets. Although transforming growth factor-ß (TGF-ß) is a primary regulator of fibrosis development, the cellular and molecular mechanisms that trigger its activation after cardiac injury remain poorly understood. Different types of anti-TGF-ß drugs have been tested for the treatment of cardiac fibrosis and have been associated with side effects. Therefore, a better understanding of these mechanisms is of great clinical relevance and could allow us to identify new therapeutic targets. Interestingly, it has been shown that platelets infiltrate the myocardium at an early stage after cardiac injury, producing large amounts of cytokines and growth factors. These molecules can directly or indirectly regulate cells involved in the fibrotic response, including cardiac fibroblasts and immune cells. In particular, platelets are known to be a major source of TGF-ß1. In this review, we have provided an overview of the classical cellular effectors involved in the pathogenesis of cardiac fibrosis, focusing on the emergent role of platelets, while discussing opportunities for novel therapeutic interventions.
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Células Endoteliais , Insuficiência Cardíaca , Humanos , Células Endoteliais/metabolismo , Miocárdio/metabolismo , Fibroblastos/metabolismo , Fibrose , Insuficiência Cardíaca/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Miócitos Cardíacos/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Platelet inhibition is the main treatment strategy to prevent atherothrombotic complications after acute coronary syndrome or percutaneous coronary intervention. Despite dual antiplatelet therapy (DAPT) combining aspirin and a P2Y12 receptor inhibitor, high on-treatment platelet reactivity (HPR) persists in some patients due to poor response to treatment and is associated with ischemic risk. Tubulin acetylation has been pointed out as a hallmark of stable microtubules responsible for the discoid shape of resting platelets. However, the impact of antiplatelet treatments on this post-translational modification has never been studied. This study investigated whether tubulin acetylation differs according to antiplatelet therapy and on-treatment platelet reactivity. Platelets were isolated from arterial blood samples of 240 patients admitted for coronary angiography, and levels of α-tubulin acetylation on lysine 40 (α-tubulin K40 acetylation) were assessed by western blot. We show that platelet α-tubulin K40 acetylation was significantly increased in DAPT-treated patients. In addition, the proportion of patients with high levels of α-tubulin K40 acetylation was drastically reduced among DAPT-treated patients with HPR. Multivariate logistic regression confirmed that DAPT resulting in adequate platelet inhibition was strongly associated with elevated α-tubulin K40 acetylation. In conclusion, our study highlights the role of elevated platelet α-tubulin K40 acetylation as a marker of platelet inhibition in response to DAPT.Clinical trial registration: https://clinicaltrials.gov - NCT03034148.
What is the context? High on-treatment platelet reactivity due to dual antiplatelet therapy poor response is associated with thrombotic risk.Acetylation of α-tubulin K40 plays a crucial role in regulating platelet shape.High α-tubulin K40 acetylation is a hallmark of stable microtubules.What is new? α-tubulin K40 acetylation is increased in platelets from dual antiplatelet therapy-treated patients.High platelet α-tubulin K40 acetylation is mainly observed in clopidogrel-responsive patients.What is the impact? Elevated acetylated K40 α-tubulin could be used as a readout of adequate platelet inhibition in response to dual antiplatelet therapy.High α-tubulin K40 acetylation could contribute to maintaining the resting morphology of circulating platelets and therefore modify their capacity to be involved in thrombotic events.
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Doença da Artéria Coronariana , Humanos , Doença da Artéria Coronariana/tratamento farmacológico , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Tubulina (Proteína) , Acetilação , Plaquetas , Processamento de Proteína Pós-TraducionalRESUMO
Heart failure (HF) with preserved ejection fraction (HFpEF) is associated with high burden of comorbidities known to increase the mean platelet volume (MPV). This parameter has been associated with morbidity and mortality in HF. However, the role of platelets and the prognostic relevance of MPV in HFpEF remain largely unexplored. We aimed to evaluate the clinical usefulness of MPV as a prognostic marker in HFpEF. We prospectively enrolled 228 patients with HFpEF (79 ± 9 years; 66% females) and 38 controls of similar age and gender (78 ± 5 years; 63% females). All subjects underwent two-dimensional echocardiography and MPV measurements. Patients were followed-up for a primary end point of all-cause mortality or first HF hospitalization. The prognostic impact of MPV was determined using Cox proportional hazard models. Mean MPV was significantly higher in HFpEF patients compared with controls (MPV: 10.7 ± 1.1fL vs. 10.1 ± 1.1fL, p = .005). HFpEF patients (n = 56) with MPV >75th percentile (11.3 fL) displayed more commonly a history of ischemic cardiomyopathy. Over a median follow-up of 26 months, 136 HFpEF patients reached the composite endpoint. MPV >75th percentile was a significant predictor of the primary endpoint (HR: 1.70 [1.08; 2.67], p = .023) adjusted for NYHA class, chronic obstructive pulmonary disease, loop diuretics, renal function, and hemoglobin. We demonstrated that MPV was significantly higher in HFpEF patients compared with controls of similar age and gender. Elevated MPV was a strong and independent predictor of poor outcome in HFpEF patients and may be relevant for clinical use.
What is the context? Heart failure with preserved ejection fraction (HFpEF) is associated with several comorbidities known to increase the mean platelet volume (MPV).MPV is a measure of platelet size and a potential marker of platelet reactivity. An increased MPV results from an increased platelet turnover.MPV has been associated with morbidity and mortality from heart failure.No study has previously compared MPV between HFpEF and controls and investigated the prognostic relevance of MPV in HFpEF disease.What is new? In this study, we compared the MPV between HFpEF patients and controls of similar age and gender, prospectively enrolled between 2015 and 2021. We evaluated the prognostic role of elevated MPV in HFpEF patients.Our main results:The MPV was higher in HFpEF patients compared to controls of similar age and gender.HFpEF patients with elevated MPV displayed more commonly a history of ischemic cardiomyopathy.Elevated MPV was a strong and independent predictor of poor outcome in HFpEF patients.What is the impact? MPV may be relevant for clinical use to predict clinical outcome in HFpEF patients.Elevated MPV reflecting platelet activity supports the potential role of platelets in HFpEF's pathophysiology.
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Insuficiência Cardíaca , Feminino , Humanos , Masculino , Insuficiência Cardíaca/diagnóstico , Prognóstico , Volume Sistólico , Volume Plaquetário Médio , Hospitalização , Função Ventricular EsquerdaRESUMO
Severe forms of coronavirus 2019 (COVID-19) disease are caused by an exaggerated systemic inflammatory response and subsequent inflammation-related coagulopathy. Anti-inflammatory treatment with low dose dexamethasone has been shown to reduce mortality in COVID-19 patients requiring oxygen therapy. However, the mechanisms of action of corticosteroids have not been extensively studied in critically ill patients in the context of COVID-19. Plasma biomarkers of inflammatory and immune responses, endothelial and platelet activation, neutrophil extracellular trap formation, and coagulopathy were compared between patients treated or not by systemic dexamethasone for severe forms of COVID-19. Dexamethasone treatment significantly reduced the inflammatory and lymphoid immune response in critical COVID-19 patients but had little effect on the myeloid immune response and no effect on endothelial activation, platelet activation, neutrophil extracellular trap formation, and coagulopathy. The benefits of low dose dexamethasone on outcome in critical COVID-19 can be partially explained by a modulation of the inflammatory response but not by reduction of coagulopathy. Future studies should explore the impact of combining dexamethasone with other immunomodulatory or anticoagulant drugs in severe COVID-19.
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COVID-19 , Citocinas , Humanos , SARS-CoV-2 , Estado Terminal , Tratamento Farmacológico da COVID-19 , COVID-19/complicações , Dexametasona/farmacologia , Dexametasona/uso terapêuticoRESUMO
Fatty acids (FAs) rapidly and efficiently reduce cardiac glucose uptake in the Randle cycle or glucose-FA cycle. This fine-tuned physiological regulation is critical to allow optimal substrate allocation during fasted and fed states. However, the mechanisms involved in the direct FA-mediated control of glucose transport have not been totally elucidated yet. We previously reported that leucine and ketone bodies, other cardiac substrates, impair glucose uptake by increasing global protein acetylation from acetyl-CoA. As FAs generate acetyl-CoA as well, we postulated that protein acetylation is enhanced by FAs and participates in their inhibitory action on cardiac glucose uptake. Here, we demonstrated that both palmitate and oleate promoted a rapid increase in protein acetylation in primary cultured adult rat cardiomyocytes, which correlated with an inhibition of insulin-stimulated glucose uptake. This glucose absorption deficit was caused by an impairment in the translocation of vesicles containing the glucose transporter GLUT4 to the plasma membrane, although insulin signaling remained unaffected. Interestingly, pharmacological inhibition of lysine acetyltransferases (KATs) prevented this increase in protein acetylation and glucose uptake inhibition induced by FAs. Similarly, FA-mediated inhibition of insulin-stimulated glucose uptake could be prevented by KAT inhibitors in perfused hearts. To summarize, enhanced protein acetylation can be considered as an early event in the FA-induced inhibition of glucose transport in the heart, explaining part of the Randle cycle.NEW & NOTEWORTHY Our results show that cardiac metabolic overload by oleate or palmitate leads to increased protein acetylation inhibiting GLUT4 translocation to the plasma membrane and glucose uptake. This observation suggests an additional regulation mechanism in the physiological glucose-FA cycle originally discovered by Randle.
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Ácidos Graxos , Ácido Oleico , Ratos , Animais , Ácidos Graxos/metabolismo , Transporte Proteico , Ácido Oleico/metabolismo , Acetilação , Acetilcoenzima A/metabolismo , Transporte Biológico , Miócitos Cardíacos/metabolismo , Glucose/metabolismo , Insulina/farmacologia , Insulina/metabolismo , Palmitatos/farmacologia , Transportador de Glucose Tipo 4/metabolismoRESUMO
Pulmonary hypertension (PH) is a chronic disorder of the pulmonary circulation that often associates with other respiratory diseases (i.e., group III PH), leading to worsened symptoms and prognosis, notably when combined with interstitial lung diseases such as pulmonary fibrosis (PF). PH may lead to right ventricular (RV) failure, which accounts for a substantial part of the mortality in chronic lung disease patients. The disappointing results of pulmonary arterial hypertension (PAH)-related therapies in patients with PF emphasize the need to better understand the pathophysiologic mechanisms that drive PH development and progression in this specific setting. In this work, we validated an animal model of group III PH associated with PF (PH-PF), by using bleomycin (BM) intratracheal instillation and characterizing the nature of induced lung and vascular remodeling, including the influence on RV structure and function. To our knowledge, this is the first work describing this dose of BM in Sprague Dawley rats and the effects upon the heart and lungs, using different techniques such as echocardiography, heart catheterization, and histology. Our data shows the successful implementation of a rat model that mimics combined PF-PH, with most features seen in the equivalent human disease, such as lung and arterial remodeling, increased mPAP and RV dysfunction.
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Pulmonary fibrosis (PF) and pulmonary hypertension (PH) are chronic diseases of the pulmonary parenchyma and circulation, respectively, which may coexist, but underlying mechanisms remain elusive. Mutations in the GCN2 (general control nonderepressible 2) gene (EIF2AK4 [eukaryotic translation initiation factor 2 alpha kinase 4]) were recently associated with pulmonary veno-occlusive disease. The aim of this study is to explore the involvement of the GCN2/eIF2α (eukaryotic initiation factor 2α) pathway in the development of PH during PF, in both human disease and in a laboratory animal model. Lung tissue from patients with PF with or without PH was collected at the time of lung transplantation, and control tissue was obtained from tumor resection surgery. Experimental lung disease was induced in either male wild-type or EIF2AK4-mutated Sprague-Dawley rats, randomly receiving a single intratracheal instillation of bleomycin or saline. Hemodynamic studies and organ collection were performed 3 weeks after instillation. Only significant results (P < 0.05) are presented. In PF lung tissue, GCN2 protein expression was decreased compared with control tissue. GCN2 expression was reduced in CD31+ endothelial cells. In line with human data, GCN2 protein expression was decreased in the lung of bleomycin rats compared with saline. EIF2AK4-mutated rats treated with bleomycin showed increased parenchymal fibrosis (hydroxyproline concentrations) and vascular remodeling (media wall thickness) as well as increased right ventricular systolic pressure compared with wild-type animals. Our data show that GCN2 is dysregulated in both humans and in an animal model of combined PF and PH. The possibility of a causative implication of GCN2 dysregulation in PF and/or PH development should be further studied.
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Hipertensão Pulmonar , Fibrose Pulmonar , Animais , Humanos , Masculino , Ratos , Bleomicina , Células Endoteliais/patologia , Hipertensão Pulmonar/patologia , Pulmão/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Fibrose Pulmonar/patologia , Ratos Sprague-DawleyRESUMO
Background: Inflammation and oxidative stress are thought to play an important role in the pathophysiology of heart failure with preserved ejection fraction (HFpEF) through the development of endothelial dysfunction. Myeloperoxidase (MPO) functions as a link between oxidative stress and inflammation and is an interesting therapeutic target. The objective of this observational cohort study was to compare MPO levels between HFpEF and old controls, to define clinical characteristics associated with high levels of MPO and to assess the relation between MPO levels and vascular function. Methods: Patients with HFpEF (N = 55) and controls > 60 years (N = 18) were prospectively included. All subjects underwent complete echocardiography and blood sampling. MPO levels were dosed by ELISA assay. Effective arterial elastance (Ea) and peripheral arterial tonometry (EndoPAT reactive hyperemia index RHI and augmentation index AIx) were used to assess vascular function. Characteristics between groups defined by the median of MPO were compared using independent samples t-test or chi square test. Results: Patients with HFpEF (80 ± 8.7 years, 65% female) had higher levels of MPO compared to controls (75 ± 5.0 years, 72% female) (34.7 ng/mL [22.7; 44.0] vs 22.6 [18.2; 32.0], p = 0.026). MPO levels were correlated with markers of inflammation; C-reactive protein (Pearson's R = 0.46, p = 0.001) and neutrophile to lymphocyte ratio (R = 0.36, p = 0.031) and with signs of left ventricular (LV) remodelling and elevated filling pressures, namely NT-proBNP levels (R = 0.32, p = 0.019), decreased LV ejection fraction (LVEF, R = -0.36, p = 0.008) and E/e' ratio (R = 0.35, p = 0.011). HFpEF patients with levels of MPO above the median were more often men (48% vs 21%, p = 0.037) and suffered more often from diabetes (48% vs 18%, p = 0.017). Intriguingly, they had lower indices of vascular stiffness (augmentation index 11.1 [0.1; 30.7] vs 19.9 [10.5; 33.4], p = 0.018 and arterial elastance Ea 2.06 ± 0.676 vs 2.43 ± 0.721, p = 0.065) and there was no difference in endothelial function (1.82 [1.34; 2.30] vs 1.66 [1.32; 1.95], p = 0.55). Conclusions: HFpEF patients have higher levels of MPO than controls, reflecting leukocyte activation and oxidative stress. Among patients, high levels of MPO are associated with male sex, diabetic status, subtle left ventricular dysfunction and pronounced diastolic dysfunction. The association between oxidative stress and vascular stiffness, on the other hand could not be demonstrated. Clinical Trial Registration: Clinical trial NCT03197350.
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Biological tissues comprise a spatially complex structure, composition and organization at the microscale, named the microstructure. Given the close structure-function relationships in tissues, structural characterization is essential to fully understand the functioning of healthy and pathological tissues, as well as the impact of possible treatments. Here, we present a nondestructive imaging approach to perform quantitative 3D histo(patho)logy of biological tissues, termed Cryogenic Contrast-Enhanced MicroCT (cryo-CECT). By combining sample staining, using an X-ray contrast-enhancing staining agent, with freezing the sample at the optimal freezing rate, cryo-CECT enables 3D visualization and structural analysis of individual tissue constituents, such as muscle and collagen fibers. We applied cryo-CECT on murine hearts subjected to pressure overload following transverse aortic constriction surgery. Cryo-CECT allowed to analyze, in an unprecedented manner, the orientation and diameter of the individual muscle fibers in the entire heart, as well as the 3D localization of fibrotic regions within the myocardial layers. We foresee further applications of cryo-CECT in the optimization of tissue/food preservation and donor banking, showing that cryo-CECT also has clinical and industrial potential.
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Sistema Musculoesquelético , Camundongos , Animais , Microtomografia por Raio-X/métodos , Congelamento , Coloração e Rotulagem , Colágeno , Imageamento Tridimensional/métodosRESUMO
Background: Neutrophil extracellular traps' (NETs') formation is a mechanism of defense that neutrophils deploy as an alternative to phagocytosis, to constrain the spread of microorganisms. Aim: The aim was to evaluate biomarkers of NETs' formation in a patient cohort admitted to intensive care unit (ICU) due to infection. Methods: Forty-six septic shock patients, 22 critical COVID-19 patients and 48 matched control subjects were recruited. Intact nucleosomes containing histone 3.1 (Nu.H3.1), or citrullinated histone H3R8 (Nu.Cit-H3R8), free citrullinated histone (Cit-H3), neutrophil elastase (NE) and myeloperoxidase (MPO) were measured. Results: Significant differences in Nu.H3.1 and NE levels were observed between septic shock and critical COVID-19 subjects as well as with controls (p-values < 0.05). The normalization of nucleosome levels according to the neutrophil count improved the discrimination between septic shock and critical COVID-19 patients. The ratio of Nu.Cit-H3R8 to Nu.H3.1 allowed the determination of nucleosome citrullination degree, presumably by PAD4. Conclusions: H3.1 and Cit-H3R8 nucleosomes appear to be interesting markers of global cell death and neutrophil activation when combined. Nu.H3.1 permits the evaluation of disease severity and differs between septic shock and critical COVID-19 patients, reflecting two distinct potential pathological processes in these conditions.
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COVID-19 , Armadilhas Extracelulares , Choque Séptico , Biomarcadores/metabolismo , Armadilhas Extracelulares/metabolismo , Histonas/metabolismo , Humanos , Neutrófilos/metabolismo , Nucleossomos/metabolismo , Choque Séptico/metabolismoRESUMO
STUDY QUESTION: What biological processes are linked to the signaling of the energy sensor 5'-AMP-activated protein kinase (AMPK) in mouse and human granulosa cells (GCs)? SUMMARY ANSWER: The lack of α1AMPK in GCs impacted cell cycle, adhesion, lipid metabolism and induced a hyperandrogenic response. WHAT IS KNOWN ALREADY: AMPK is expressed in the ovarian follicle, and its activation by pharmacological medications, such as metformin, inhibits the production of steroids. Polycystic ovary syndrome (PCOS) is responsible for infertility in approximately 5-20% of women of childbearing age and possible treatments include reducing body weight, improving lifestyle and the administration of a combination of drugs to improve insulin resistance, such as metformin. STUDY DESIGN, SIZE, DURATION: AMPK signaling was evaluated by analyzing differential gene expression in immortalized human granulosa cells (KGNs) with and without silencing α1AMPK using CRISPR/Cas9. In vivo studies included the use of a α1AMPK knock-out mouse model to evaluate the role of α1AMPK in folliculogenesis and fertility. Expression of α1AMPK was evaluated in primary human granulosa-luteal cells retrieved from women undergoing IVF with and without a lean PCOS phenotype (i.e. BMI: 18-25 kg/m2). PARTICIPANTS/MATERIALS, SETTING, METHODS: α1AMPK was disrupted in KGN cells and a transgenic mouse model. Cell viability, proliferation and metabolism were evaluated. Androgen production was evaluated by analyzing protein levels of relevant enzymes in the steroid pathway by western blots, and steroid levels obtained from in vitro and in vivo models by mass spectrometry. Differential gene expression in human GC was obtained by RNA sequencing. Analysis of in vivo murine folliculogenesis was performed by histology and immunochemistry, including evaluation of the anti-Müllerian hormone (AMH) marker. The α1AMPK gene expression was evaluated by quantitative RT-PCR in primary GCs obtained from women with the lean PCOS phenotype (n = 8) and without PCOS (n = 9). MAIN RESULTS AND THE ROLE OF CHANCE: Silencing of α1AMPK in KGN increased cell proliferation (P < 0.05 versus control, n = 4), promoted the use of fatty acids over glucose, and induced a hyperandrogenic response resulting from upregulation of two of the enzymes involved in steroid production, namely 3ß-hydroxysteroid dehydrogenase (3ßHSD) and P450 side-chain cleavage enzyme (P450scc) (P < 0.05, n = 3). Female mice deficient in α1AMPK had a 30% decrease in their ovulation rate (P < 0.05, n = 7) and litter size, a hyperandrogenic response (P < 0.05, n = 7) with higher levels of 3ßHSD and p450scc levels in the ovaries, and an increase in the population of antral follicles (P < 0.01, n = 10) compared to controls. Primary GCs from lean women with PCOS had lower α1AMPK mRNA expression levels than the control group (P < 0.05, n = 8-9). LARGE SCALE DATA: The FastQ files and metadata were submitted to the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB46048. LIMITATIONS, REASONS FOR CAUTION: The human KGN is a not fully differentiated, transformed cell line. As such, to confirm the role of AMPK in GC and the PCOS phenotype, this model was compared to two others: an α1AMPK transgenic mouse model and primary differentiated granulosa-lutein cells from non-obese women undergoing IVF (with and without PCOS). A clear limitation is the small number of patients with PCOS utilized in this study and that the collection of human GCs was performed after hormonal stimulation. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal that AMPK is directly involved in steroid production in human GCs. In addition, AMPK signaling was associated with other processes frequently reported as dysfunctional in PCOS models, such as cell adhesion, lipid metabolism and inflammation. Silencing of α1AMPK in KGN promoted folliculogenesis, with increases in AMH. Evaluating the expression of the α1AMPK subunit could be considered as a marker of interest in infertility cases related to hormonal imbalances and metabolic disorders, including PCOS. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by the Institut National de la Recherche Agronomique (INRA) and the national programme « FERTiNERGY ¼ funded by the French National Research Agency (ANR). The authors report no intellectual or financial conflicts of interest related to this work. R.K. is identified as personnel of the International Agency for Research on Cancer/World Health Organization. R.K. alone is responsible for the views expressed in this article and she does not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization. TRIAL REGISTRATION NUMBER: N/A.
Assuntos
Fenômenos Biológicos , Hiperandrogenismo , Infertilidade Feminina , Metformina , Síndrome do Ovário Policístico , Proteínas Quinases Ativadas por AMP , Animais , Hormônio Antimülleriano/metabolismo , Feminino , Fertilidade , Humanos , Hiperandrogenismo/complicações , Metformina/farmacologia , Camundongos , Síndrome do Ovário Policístico/metabolismoRESUMO
Our group previously demonstrated that an excess of nutrients, as observed in diabetes, provokes an increase in cardiac protein acetylation responsible for a reduced insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The acetylated proteins involved in this event have yet not been identified. α-Tubulin is a promising candidate as a major cytoskeleton component involved, among other things, in the translocation of GLUT4-containing vesicles from their intracellular pools toward the plasma membrane. Moreover, α-tubulin is known to be acetylated, Lys40 (K40) being its best characterized acetylated residue. The present work sought to evaluate the impact of α-tubulin K40 acetylation on cardiac glucose entry, with a particular interest in GLUT4 translocation. First, we observed that a mouse model of high-fat diet-induced obesity presented an increase in cardiac α-tubulin K40 acetylation level. We next showed that treatment of insulin-sensitive primary cultured adult rat cardiomyocytes with tubacin, a specific tubulin acetylation inducer, reduced insulin-stimulated glucose uptake and GLUT4 translocation. Conversely, decreasing α-tubulin K40 acetylation by expressing a nonacetylable dominant form of α-tubulin (mCherry α-tubulin K40A mutant) remarkably intensified insulin-induced glucose transport. Finally, mCherry α-tubulin K40A expression similarly improved glucose transport in insulin-resistant cardiomyocytes or after AMP-activated protein kinase activation. Taken together, our study demonstrates that modulation of α-tubulin K40 acetylation level affects glucose transport in cardiomyocytes, offering new putative therapeutic insights regarding modulation of glucose metabolism in insulin-resistant and diabetic hearts.NEW & NOTEWORTHY Acetylation level of α-tubulin on K40 is increased in the heart of a diet-induced mouse model of type 2 diabetes. Pharmacological stimulation of α-tubulin K40 acetylation lowers insulin-mediated GLUT4 vesicles translocation to the plasma membrane, reducing glucose transport. Expressing a nonacetylable dominant form of α-tubulin boosts glucose uptake in both insulin-sensitive and insulin-resistant cardiomyocytes.
Assuntos
Diabetes Mellitus Tipo 2 , Glucose , Miócitos Cardíacos , Tubulina (Proteína) , Proteínas Quinases Ativadas por AMP/metabolismo , Acetilação , Animais , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Lisina/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Transporte Proteico , Ratos , Tubulina (Proteína)/metabolismoRESUMO
Protein O-GlcNAcylation is increasingly recognized as an important cellular regulatory mechanism, in multiple organs including the heart. However, the mechanisms leading to O-GlcNAcylation in mitochondria and the consequences on their function remain poorly understood. In this study, we use an in vitro reconstitution assay to characterize the intra-mitochondrial O-GlcNAc system without potential cytoplasmic confounding effects. We compare the O-GlcNAcylome of isolated cardiac mitochondria with that of mitochondria acutely exposed to NButGT, a specific inhibitor of glycoside hydrolase. Amongst the 409 O-GlcNAcylated mitochondrial proteins identified, 191 display increased O-GlcNAcylation in response to NButGT. This is associated with enhanced Complex I (CI) activity, increased maximal respiration in presence of pyruvate-malate, and a striking reduction of mitochondrial ROS release, which could be related to O-GlcNAcylation of specific subunits of ETC complexes (CI, CIII) and TCA cycle enzymes. In conclusion, our work underlines the existence of a dynamic mitochondrial O-GlcNAcylation system capable of rapidly modifying mitochondrial function.
Assuntos
Acetilglucosamina , Mitocôndrias Cardíacas , Coração , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismoAssuntos
Cardiomegalia , Interleucina-18 , Animais , Cardiomegalia/genética , Cardiomegalia/metabolismo , Fibrose , Expressão Gênica , CamundongosRESUMO
The platelet transmembrane receptor GPVI can be assessed together with other platelet membrane markers in a whole blood multicolor flow cytometry panel. The advantage of combining multiple antibodies in a single tube is the possibility of distinguishing multiple platelet subgroups. In this short communication, we describe an activation problem encountered with anti-GPVI, clone HY101. Activation of platelets was seen after the addition of anti-GPVI in a flow cytometry panel, highlighted by the expression of the activation markers CD62P, PAC-1, CD63, and CD107a. This was also confirmed by platelet aggregation studies.
Assuntos
Plaquetas , Glicoproteínas da Membrana de Plaquetas , Plaquetas/metabolismo , Citometria de Fluxo , Humanos , Ativação Plaquetária , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismoRESUMO
Acetyl-CoA carboxylase (ACC) is the first enzyme regulating de novo lipid synthesis via the carboxylation of acetyl-CoA into malonyl-CoA. The inhibition of its activity decreases lipogenesis and, in parallel, increases the acetyl-CoA content, which serves as a substrate for protein acetylation. Several findings support a role for acetylation signaling in coordinating signaling systems that drive platelet cytoskeletal changes and aggregation. Therefore, we investigated the impact of ACC inhibition on tubulin acetylation and platelet functions. Human platelets were incubated 2 h with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. We have herein demonstrated that CP640.186 treatment does not affect overall platelet lipid content, yet it is associated with increased tubulin acetylation levels, both at the basal state and after thrombin stimulation. This resulted in impaired platelet aggregation. Similar results were obtained using human platelets that were pretreated with tubacin, an inhibitor of tubulin deacetylase HDAC6. In addition, both ACC and HDAC6 inhibitions block key platelet cytoskeleton signaling events, including Rac1 GTPase activation and the phosphorylation of its downstream effector, p21-activated kinase 2 (PAK2). However, neither CP640.186 nor tubacin affects thrombin-induced actin cytoskeleton remodeling, while ACC inhibition results in decreased thrombin-induced reactive oxygen species (ROS) production and extracellular signal-regulated kinase (ERK) phosphorylation. We conclude that when using washed human platelets, ACC inhibition limits tubulin deacetylation upon thrombin stimulation, which in turn impairs platelet aggregation. The mechanism involves a downregulation of the Rac1/PAK2 pathway, being independent of actin cytoskeleton.