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RATIONALE: Heavy-labelled internal standards increasingly represent the gold standard for absolute quantitation in mass spectrometry (MS)-based bottom-up proteomics. The biggest drawbacks of using these standards are that they have high costs and lengthy lead times. METHODS: We describe an efficient, low-cost optimised method to enable 'in-house' heavy labelling of synthetic tryptic peptides for absolute quantification using tandem LC-MS/MS mass spectrometry. Our methodology uses 18O water in a trypsin-catalysed oxygen exchange reaction at the carboxyl terminus with the overall aim of reducing the costs and lead time associated with sourcing heavy standards from commercial vendors. RESULTS: Step-by-step instructions are provided on how to execute this protocol with high-throughput adaptations utilising a 96-well plate and a liquid-handling robot. Detailed notes on experimental setup, tips for troubleshooting and suggested improvements to maximise labelling efficiencies are highlighted to achieve the best results. Under optimum conditions, labelling efficiencies of peptides can reach from 95% to 100%. CONCLUSIONS: The application of the 'in-house' labelled standards in generating calibration curves to quantify endogenous peptide concentrations is just as effective as using the synthetically sourced standards while also having great cost reduction implications as well as saving time spent waiting for peptides to arrive. The protocol is highly adaptable and can be customized to fit the specific setup of any laboratory, maximizing achievable labelling efficiencies.

Asunto(s)

Marcaje Isotópico , Péptidos , Proteómica , Espectrometría de Masas en Tándem , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Péptidos/química , Péptidos/análisis , Marcaje Isotópico/métodos , Isótopos de Oxígeno/análisis , Isótopos de Oxígeno/química , Tripsina/química , Tripsina/metabolismo , Cromatografía Liquida/métodos

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AIMS: We investigated the prevalence, clinical characteristics, and prognosis of patients with heart failure (HF) with improved ejection fraction (HFimpEF). METHODS AND RESULTS: We used data from BIOSTAT-CHF including patients with a left ventricular ejection fraction (LVEF) ≤40% at baseline who had LVEF re-assessed at 9 months. HFimpEF was defined as a LVEF >40% and a LVEF ≥10% increase from baseline at 9 months. We validated findings in the ASIAN-HF registry. The primary outcome was a composite of time to HF rehospitalization or all-cause mortality. In BIOSTAT-CHF, about 20% of patients developed HFimpEF, that was associated with a lower primary event rate of all-cause mortality (hazard ratio [HR] 0.52, 95% confidence interval [CI] 0.28-0.97, p = 0.040) and the composite endpoint (HR 0.46, 95% CI 0.30-0.70, p < 0.001) compared with patients who remained in persistent HF with reduced ejection fraction (HFrEF). The findings were similar in the ASIAN-HF (HR 0.40, 95% CI 0.18-0.89, p = 0.024, and HR 0.29, 95% CI 0.17-0.48, p < 0.001). Five independently common predictors for HFimpEF in both BIOSTAT-CHF and ASIAN-HF were female sex, absence of ischaemic heart disease, higher LVEF, smaller left ventricular end-diastolic and end-systolic diameter at baseline. A predictive model combining only five predictors (absence of ischaemic heart disease and left bundle branch block, smaller left ventricular end-systolic and left atrial diameter, and higher platelet count) for HFimpEF in the BIOSTAT-CHF achieved an area under the curve of 0.772 and 0.688 in the ASIAN-HF (due to missing left atrial diameter and platelet count). CONCLUSIONS: Approximately 20-30% of patients with HFrEF improved to HFimpEF within 1 year with better clinical outcomes. In addition, the predictive model with clinical predictors could more accurately predict HFimpEF in patients with HFrEF.

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BACKGROUND AND AIMS: Circulating proenkephalin (PENK) is a stable endogenous polypeptide with fast response to glomerular dysfunction and tubular damage. This study examined the predictive value of PENK for renal outcomes and mortality in patients with acute coronary syndromes (ACS). METHODS: Proenkephalin was measured in plasma in a prospective multicentre ACS cohort from Switzerland (n=4787) and in validation cohorts from the UK (n=1141), Czechia (n=927), and Germany (n=220). A biomarker-enhanced risk score (KID-ACS score) for simultaneous prediction of in-hospital acute kidney injury (AKI) and 30-day mortality was derived and externally validated. RESULTS: On multivariable adjustment for established risk factors, circulating PENK remained associated with in-hospital AKI (per log2 increase: adjusted odds ratio [OR] 1.53, 95% confidence interval [CI] 1.13-2.09, P=0.007) and 30-day mortality (adjusted hazard ratio [HR] 2.73, 95% CI 1.85-4.02, P<0.001). The KID-ACS score integrates PENK and showed an area under the receiver operating characteristic curve (AUC) of 0.72 (95% CI 0.68-0.76) for in-hospital AKI, and of 0.91 (95% CI 0.87-0.95) for 30-day mortality in the derivation cohort. Upon external validation, KID-ACS achieved similarly high performance for in-hospital AKI (Zurich: AUC 0.73, 95% CI 0.70-0.77; Czechia: AUC 0.75, 95% CI 0.68-0.81; Germany: AUC 0.71, 95% CI 0.55-0.87) and 30-day mortality (UK: AUC 0.87, 95% CI 0.83-0.91; Czechia: AUC 0.91, 95% CI 0.87-0.94; Germany: AUC 0.96, 95% CI 0.92-1.00) outperforming the CA-AKI score and the GRACE 2.0 score, respectively. CONCLUSIONS: Circulating PENK offers incremental value for predicting in-hospital AKI and mortality in ACS. The simple 6-item KID-ACS risk score integrates PENK and provides a novel tool for simultaneous assessment of renal and mortality risk in patients with ACS.

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BACKGROUND: Dysregulated epicardial adipose tissue (EAT) may contribute to the development of heart failure in Type 2 diabetes (T2D). This study aimed to evaluate the associations between EAT volume and composition with imaging markers of subclinical cardiac dysfunction in people with T2D and no prevalent cardiovascular disease. METHODS: Prospective case-control study enrolling participants with and without T2D and no known cardiovascular disease. Two hundred and fifteen people with T2D (median age 63 years, 60 % male) and thirty-nine non-diabetics (median age 59 years, 62 % male) were included. Using computed tomography (CT), total EAT volume and mean CT attenuation, as well as, low attenuation (Hounsfield unit range -190 to -90) EAT volume were quantified by a deep learning method and volumes indexed to body surface area. Associations with cardiac magnetic resonance-derived left ventricular (LV) volumes and strain indices were assessed using linear regression. RESULTS: T2D participants had higher LV mass/volume ratio (median 0.89 g/mL [0.82-0.99] vs 0.79 g/mL [0.75-0.89]) and lower global longitudinal strain (GLS; 16.1 ± 2.3 % vs 17.2 ± 2.2 %). Total indexed EAT volume correlated inversely with mean CT attenuation. Low attenuation indexed EAT volume was 2-fold higher (18.8 cm3/m2 vs. 9.4 cm3/m2, p < 0.001) in T2D and independently associated with LV mass/volume ratio (ß = 0.002, p = 0.01) and GLS (ß = -0.03, p = 0.03). CONCLUSIONS: Higher EAT volumes seen in T2D are associated with a lower mean CT attenuation. Low attenuation indexed EAT volume is independently, but only weakly, associated with markers of subclinical cardiac dysfunction in T2D.

Asunto(s)

Diabetes Mellitus Tipo 2 , Insuficiencia Cardíaca , Disfunción Ventricular Izquierda , Humanos , Masculino , Persona de Mediana Edad , Femenino , Tejido Adiposo Epicárdico , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/complicaciones , Pericardio/diagnóstico por imagen , Tejido Adiposo/diagnóstico por imagen , Disfunción Ventricular Izquierda/diagnóstico por imagen , Disfunción Ventricular Izquierda/etiología

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Quantitation of proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is complex, with a multiplicity of options ranging from label-free techniques to chemically and metabolically labeling proteins. Increasingly, for clinically relevant analyses, stable isotope-labeled (SIL) internal standards (ISs) represent the "gold standard" for quantitation due to their similar physiochemical properties to the analyte, wide availability, and ability to multiplex to several peptides. However, the purchase of SIL-ISs is a resource-intensive step in terms of cost and time, particularly for screening putative biomarker panels of hundreds of proteins. We demonstrate an alternative strategy utilizing nonhuman sera as the IS for quantitation of multiple human proteins. We demonstrate the effectiveness of this strategy using two high abundance clinically relevant analytes, vitamin D binding protein [Gc globulin] (DBP) and albumin (ALB). We extend this to three putative risk markers for cardiovascular disease: plasma protease C1 inhibitor (SERPING1), annexin A1 (ANXA1), and protein kinase, DNA-activated catalytic subunit (PRKDC). The results show highly specific, reproducible, and linear measurement of the proteins of interest with comparable precision and accuracy to the gold standard SIL-IS technique. This approach may not be applicable to every protein, but for many proteins it can offer a cost-effective solution to LC-MS/MS protein quantitation.

Asunto(s)

Cromatografía Líquida con Espectrometría de Masas , Espectrometría de Masas en Tándem , Animales , Humanos , Biomarcadores/sangre , Análisis Costo-Beneficio , Marcaje Isotópico/métodos , Cromatografía Líquida con Espectrometría de Masas/métodos , Péptidos/química , Péptidos/sangre , Péptidos/análisis , Proteómica/métodos , Proteómica/economía , Estándares de Referencia , Reproducibilidad de los Resultados , Albúmina Sérica/análisis , Albúmina Sérica/química , Espectrometría de Masas en Tándem/métodos , Tripsina/química , Tripsina/metabolismo , Proteína de Unión a Vitamina D/sangre , Proteína de Unión a Vitamina D/química

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BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) and atrial fibrillation (AF) frequently co-exist. There is a limited understanding on whether this coexistence is associated with distinct alterations in myocardial remodelling and mechanics. We aimed to determine if patients with atrial fibrillation (AF) and heart failure with preserved ejection fraction (HFpEF) represent a distinct phenotype. METHODS: In this secondary analysis of adults with HFpEF (NCT03050593), participants were comprehensively phenotyped with stress cardiac MRI, echocardiography and plasma fibroinflammatory biomarkers, and were followed for the composite endpoint (HF hospitalisation or death) at a median of 8.5 years. Those with AF were compared to sinus rhythm (SR) and unsupervised cluster analysis was performed to explore possible phenotypes. RESULTS: 136 subjects were included (SR = 75, AF = 61). The AF group was older (76 ± 8 vs. 70 ± 10 years) with less diabetes (36% vs. 61%) compared to the SR group and had higher left atrial (LA) volumes (61 ± 30 vs. 39 ± 15 mL/m2, p < 0.001), lower LA ejection fraction (EF) (31 ± 15 vs. 51 ± 12%, p < 0.001), worse left ventricular (LV) systolic function (LVEF 63 ± 8 vs. 68 ± 8%, p = 0.002; global longitudinal strain 13.6 ± 2.9 vs. 14.7 ± 2.4%, p = 0.003) but higher LV peak early diastolic strain rates (0.73 ± 0.28 vs. 0.53 ± 0.17 1/s, p < 0.001). The AF group had higher levels of syndecan-1, matrix metalloproteinase-2, proBNP, angiopoietin-2 and pentraxin-3, but lower level of interleukin-8. No difference in clinical outcomes was observed between the groups. Three distinct clusters were identified with the poorest outcomes (Log-rank p = 0.029) in cluster 2 (hypertensive and fibroinflammatory) which had equal representation of SR and AF. CONCLUSIONS: Presence of AF in HFpEF is associated with cardiac structural and functional changes together with altered expression of several fibro-inflammatory biomarkers. Distinct phenotypes exist in HFpEF which may have differing clinical outcomes.

Asunto(s)

Fibrilación Atrial , Insuficiencia Cardíaca , Imágenes de Resonancia Magnética Multiparamétrica , Humanos , Adulto , Volumen Sistólico , Metaloproteinasa 2 de la Matriz , Función Ventricular Izquierda , Biomarcadores , Fenotipo , Pronóstico

RESUMEN

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is a heterogenous multi-system syndrome with limited efficacious treatment options. The prevalence of Type 2 diabetes (T2D) continues to rise and predisposes patients to HFpEF, and HFpEF remains one of the biggest challenges in cardiovascular medicine today. Novel therapeutic targets are required to meet this important clinical need. Deep phenotyping studies including -OMIC analyses can provide important pathogenic information to aid the identification of such targets. The aims of this study were to determine; 1) the impact of a low-energy diet on plasma sphingolipid/ceramide profiles in people with T2D compared to healthy controls and, 2) if the change in sphingolipid/ceramide profile is associated with reverse cardiovascular remodelling. METHODS: Post-hoc analysis of a randomised controlled trial (NCT02590822) including adults with T2D with no cardiovascular disease who completed a 12-week low-energy (∼810 kcal/day) meal-replacement plan (MRP) and matched healthy controls (HC). Echocardiography, cardiac MRI and a fasting blood for lipidomics were undertaken pre/post-intervention. Candidate biomarkers were identified from case-control comparison (fold change > 1.5 and statistical significance p < 0.05) and their response to the MRP reported. Association between change in biomarkers and change indices of cardiac remodelling were explored. RESULTS: Twenty-four people with T2D (15 males, age 51.1 ± 5.7 years), and 25 HC (15 male, 48.3 ± 6.6 years) were included. Subjects with T2D had increased left ventricular (LV) mass:volume ratio (0.84 ± 0.13 vs. 0.70 ± 0.08, p < 0.001), increased systolic function but impaired diastolic function compared to HC. Twelve long-chain polyunsaturated sphingolipids, including four ceramides, were downregulated in subjects with T2D at baseline. Three sphingomyelin species and all ceramides were inversely associated with LV mass:volume. There was a significant increase in all species and shift towards HC following the MRP, however, none of these changes were associated with reverse cardiac remodelling. CONCLUSION: The lack of association between change in sphingolipids/ceramides and reverse cardiac remodelling following the MRP casts doubt on a causative role of sphingolipids/ceramides in the progression of heart failure in T2D. TRIAL REGISTRATION: NCT02590822.

Asunto(s)

Diabetes Mellitus Tipo 2 , Insuficiencia Cardíaca , Remodelación Ventricular , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Biomarcadores , Ceramidas , Ayuno , Esfingolípidos , Volumen Sistólico/fisiología , Función Ventricular Izquierda

RESUMEN

OBJECTIVES: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. METHODS: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. RESULTS: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). CONCLUSIONS: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.

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COVID-19 , Espectrometría de Masas , Técnicas de Diagnóstico Molecular , ARN Viral , SARS-CoV-2 , Saliva , Humanos , Saliva/virología , Saliva/química , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/inmunología , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/virología , ARN Viral/análisis , Espectrometría de Masas/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Masculino , Sensibilidad y Especificidad , Femenino , Persona de Mediana Edad , Fosfoproteínas/análisis , Fosfoproteínas/inmunología , Proteínas de la Nucleocápside de Coronavirus/análisis , Proteínas de la Nucleocápside de Coronavirus/inmunología , Antígenos Virales/análisis , Antígenos Virales/inmunología , Adulto , Cromatografía Liquida/métodos

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Insuficiencia Cardíaca , Infarto del Miocardio , Humanos , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/terapia , Infarto del Miocardio/diagnóstico

RESUMEN

AIMS: Heart failure (HF) is associated with cytokine activation and inflammation. Experimental evidence suggests that plasma interleukin-17 (IL-17) is associated with myocardial fibrosis and cardiac dysfunction in HF. IL-17D, a subtype of IL-17 originates from particular tissues such as the heart. However, there is very limited data on the IL-17 cytokine family in patients with HF. Therefore, we investigated the association between circulating IL-17D levels, clinical characteristics and outcome in a large cohort of patients with heart failure. METHODS AND RESULTS: Plasma IL-17D was measured in 2032 patients with HF from 11 European countries using a proximity extension assay. The primary outcome was a composite of HF hospitalization or all-cause mortality. Patients with higher plasma IL-17D concentrations were more likely to have atrial fibrillation (AF), renal dysfunction and heart failure with preserved ejection fraction (HFpEF) and had higher plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) concentrations (all p < 0.001). IL-17D was not associated with interleukin-6 (IL-6) or C-reactive protein (CRP) concentrations. After adjustment for confounders in a multivariable Cox regression analysis, patients in the highest quartile of plasma IL-17D had a significantly increased risk of the composite outcome of HF hospitalization or all-cause mortality compared to patients in the lowest quartile [Hazard ratio (HR) 1.28, 95% confidence interval (CI) 1.05-1.57]. CONCLUSION: In patients with HF, elevated plasma IL-17D concentrations are associated with higher plasma NT-proBNP concentrations and a higher prevalence of AF and renal dysfunction. High IL-17D concentrations are independently associated with worse outcome.

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Insuficiencia Cardíaca , Interleucina-27 , Enfermedades Renales , Humanos , Interleucina-17 , Volumen Sistólico/fisiología , Pronóstico , Péptido Natriurético Encefálico , Fragmentos de Péptidos , Biomarcadores

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BACKGROUND: Recent data suggest different causes of renal dysfunction between heart failure with reduced (HFrEF) versus preserved ejection fraction (HFpEF). We therefore studied a wide range of urinary markers reflecting different nephron segments in heart failure patients. METHODS: In 2070, in chronic heart failure patients, we measured several established and upcoming urinary markers reflecting different nephron segments. RESULTS: Mean age was 70 ± 12 years, 74% was male and 81% (n = 1677) had HFrEF. Mean estimated glomerular filtration rate (eGFR) was lower in patients with HFpEF (56 ± 23 versus 63 ± 23 ml/min/1.73 m2, P = 0.001). Patients with HFpEF had significantly higher values of NGAL (58.1 [24.0-124.8] versus 28.1 [14.6-66.9] µg/gCr, P < 0.001) and KIM-1 (2.28 [1.49-4.37] versus 1.79 [0.85-3.49] µg/gCr, P = 0.001). These differences were more pronounced in patients with an eGFR > 60 ml/min/1.73m2. CONCLUSIONS: HFpEF patients showed more evidence of tubular damage and/or dysfunction compared with HFrEF patients, in particular when glomerular function was preserved.

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Insuficiencia Cardíaca , Humanos , Masculino , Persona de Mediana Edad , Anciano , Anciano de 80 o más Años , Insuficiencia Cardíaca/diagnóstico , Volumen Sistólico , Enfermedad Crónica , Tasa de Filtración Glomerular , Pronóstico

RESUMEN

BACKGROUND: Mitral regurgitation (MR) frequently coexists with heart failure (HF). OBJECTIVES: To better understand potential pathophysiological differences between patients with HF with or without moderate-severe MR, we compared differentially expressed circulating biomarkers between these two groups. METHODS: The Olink Proteomics® Multiplex Cardiovascular (CVD) -II, CVD-III, Immune Response and Oncology-II panels of 363 unique proteins from different pathophysiological domains were used to investigate the biomarker profiles of HF patients from index and validation cohorts of the BIOSTAT-CHF study stratified according to the presence of moderate-to-severe MR or no-mild MR. RESULTS: The index cohort included 888 patients (46%) with moderate-to-severe MR and 1029 (54%) with no-mild MR at baseline. The validation cohort included 522 patients (33%) with moderate-to-severe MR and 1076 (66%) with no-mild MR at baseline. Compared to patients with no-mild MR, those with moderate-to-severe MR had lower body mass index, higher comorbidity burden, signs and symptoms of more severe HF, lower systolic blood pressure, and larger left atrial and ventricular dimensions, in both cohorts. NT-proBNP, CA125, fibroblast growth factor 23 (FGF23) and growth hormone 1 (GH1) were up-regulated, whereas leptin (LEP) was down-regulated in patients with moderate-severe MR versus no-mild MR, in both index and validation cohorts. CONCLUSION: Circulating biomarkers differently expressed in HF patients with moderate-severe MR versus no-mild MR were related to congestion, lipid and mineral metabolism and oxidative stress. These findings may be of value for the development of novel treatment targets in HF patients with MR.

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Insuficiencia Cardíaca , Insuficiencia de la Válvula Mitral , Humanos , Insuficiencia de la Válvula Mitral/diagnóstico por imagen , Biomarcadores , Atrios Cardíacos , Ventrículos Cardíacos

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BACKGROUND: Proenkephalin A 119-159 (PENK) is freely filtered in the glomerulus with plasma levels correlating with glomerular filtration rate. Therefore, PENK has been proposed as an early indicator of acute kidney injury (AKI) although its performance is dependent on the clinical setting. This meta-analysis aimed to investigate the correlation between PENK levels and the development of AKI. METHODS: We conducted a comprehensive search on the PubMed, Embase, Cochrane databases, the website ClinicalTrials.gov and Cnki.net until June 26, 2023. Summary receiver operating characteristic (SROC) curves were used to amalgamate the overall test performance. Diagnostic odds ratio (DOR) was employed to compare the diagnostic accuracy of PENK with other biomarkers. Quality of the evidence was assessed using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) criteria. RESULTS: We incorporated 11 observational studies with 3969 patients with an incidence of AKI of 23.4% (929 out of 3969 patients) with the best optimal cutoff value of PENK for early detection of AKI being 57.3 pmol/L. The overall sensitivity and specificity of PENK in identifying AKI were 0.69 (95% CI 0.62-0.75) and 0.76 (95% CI 0.68-0.82), respectively. The combined positive likelihood ratio (LR) stood at 2.83 (95% CI 2.06-3.88), and the negative LR was 0.41 (95% CI 0.33-0.52). The SROC curve showcased pooled diagnostic accuracy of 0.77 (95% CI 0.73-0.81). Interestingly, patients with a history of hypertension or heart failure demonstrated a lower specificity of PENK in correlating the development of AKI. CONCLUSION: Our results indicate that PENK possesses significant potential as a biomarker for the early detection of the development of AKI, using a cutoff point of 57.3 pmol/L for PENK.

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Lesión Renal Aguda , Insuficiencia Cardíaca , Humanos , Biomarcadores , Lesión Renal Aguda/diagnóstico , Tasa de Filtración Glomerular

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BACKGROUND: Despite major advances in pharmacological treatment for patients with heart failure, residual mortality remains high. This suggests that important pathways are not yet targeted by current heart failure therapies. OBJECTIVES: We sought integration of genetic, transcriptomic, and proteomic data in a large cohort of patients with heart failure to detect major pathways related to progression of heart failure leading to death. METHODS: We used machine learning methodology based on stacked generalization framework and gradient boosting algorithms, using 54 clinical phenotypes, 403 circulating plasma proteins, 36,046 transcript expression levels in whole blood, and 6 million genomic markers to model all-cause mortality in 2,516 patients with heart failure from the BIOSTAT-CHF (Systems BIOlogy Study to TAilored Treatment in Chronic Heart Failure) study. Results were validated in an independent cohort of 1,738 patients. RESULTS: The mean age of the patients was 70 years (Q1-Q3: 61-78 years), 27% were female, median N-terminal pro-B-type natriuretic peptide was 4,275 ng/L (Q1-Q3: 2,360-8,486 ng/L), and 7% had heart failure with preserved ejection fraction. During a median follow-up of 21 months, 657 (26%) of patients died. The 4 major pathways with a significant association to all-cause mortality were: 1) the PI3K/Akt pathway; 2) the MAPK pathway; 3) the Ras signaling pathway; and 4) epidermal growth factor receptor tyrosine kinase inhibitor resistance. Results were validated in an independent cohort of 1,738 patients. CONCLUSIONS: A systems biology approach integrating genomic, transcriptomic, and proteomic data identified 4 major pathways related to mortality. These pathways are related to decreased activation of the cardioprotective ERBB2 receptor, which can be modified by neuregulin.

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Insuficiencia Cardíaca , Proteómica , Humanos , Femenino , Anciano , Masculino , Biomarcadores , Multiómica , Fosfatidilinositol 3-Quinasas/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico

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OBJECTIVE: To characterise cardiac remodelling, exercise capacity and fibroinflammatory biomarkers in patients with aortic stenosis (AS) with and without diabetes, and assess the impact of diabetes on outcomes. METHODS: Patients with moderate or severe AS with and without diabetes underwent echocardiography, stress cardiovascular magnetic resonance (CMR), cardiopulmonary exercise testing and plasma biomarker analysis. Primary endpoint for survival analysis was a composite of cardiovascular mortality, myocardial infarction, hospitalisation with heart failure, syncope or arrhythmia. Secondary endpoint was all-cause death. RESULTS: Diabetes (n=56) and non-diabetes groups (n=198) were well matched for age, sex, ethnicity, blood pressure and severity of AS. The diabetes group had higher body mass index, lower estimated glomerular filtration rate and higher rates of hypertension, hyperlipidaemia and symptoms of AS. Biventricular volumes and systolic function were similar, but the diabetes group had higher extracellular volume fraction (25.9%±3.1% vs 24.8%±2.4%, p=0.020), lower myocardial perfusion reserve (2.02±0.75 vs 2.34±0.68, p=0.046) and lower percentage predicted peak oxygen consumption (68%±21% vs 77%±17%, p=0.002) compared with the non-diabetes group. Higher levels of renin (log10renin: 3.27±0.59 vs 2.82±0.69 pg/mL, p<0.001) were found in diabetes. Multivariable Cox regression analysis showed diabetes was not associated with cardiovascular outcomes, but was independently associated with all-cause mortality (HR 2.04, 95% CI 1.05 to 4.00; p=0.037). CONCLUSIONS: In patients with moderate-to-severe AS, diabetes is associated with reduced exercise capacity, increased diffuse myocardial fibrosis and microvascular dysfunction, but not cardiovascular events despite a small increase in mortality.

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Estenosis de la Válvula Aórtica , Diabetes Mellitus , Humanos , Tolerancia al Ejercicio , Renina , Estenosis de la Válvula Aórtica/diagnóstico por imagen , Corazón

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In order for mass spectrometry to continue to grow as a platform for high-throughput clinical and translational research, careful consideration must be given to quality control by ensuring that the assay performs reproducibly and accurately and precisely. In particular, the throughput required for large cohort clinical validation in biomarker discovery and diagnostic screening has driven the growth of multiplexed targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) assays paired with sample preparation and analysis in multiwell plates. However, large scale MS-based proteomics studies are often plagued by batch effects: sources of technical variation in the data, which can arise from a diverse array of sources such as sample preparation batches, different reagent lots, or indeed MS signal drift. These batch effects can confound the detection of true signal differences, resulting in incorrect conclusions being drawn about significant biological effects or lack thereof. Here, we present an intraplate batch effect termed the edge effect arising from temperature gradients in multiwell plates, commonly reported in preclinical cell culture studies but not yet reported in a clinical proteomics setting. We present methods herein to ameliorate the phenomenon including proper assessment of heating techniques for multiwell plates and incorporation of surrogate standards, which can normalize for intraplate variation.

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Proteómica , Espectrometría de Masas en Tándem , Humanos , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Proteómica/métodos , Control de Calidad , Estándares de Referencia

RESUMEN

AIMS: The importance of autoantibodies (AABs) against adrenergic/muscarinic receptors in heart failure (HF) is not well-understood. We investigated the prevalence and clinical/prognostic associations of four AABs recognizing the M2-muscarinic receptor or the ß1-, ß2-, or ß3-adrenergic receptor in a large and well-characterized cohort of patients with HF. METHODS AND RESULTS: Serum samples from 2256 patients with HF from the BIOSTAT-CHF cohort and 299 healthy controls were analysed using newly established chemiluminescence immunoassays. The primary outcome was a composite of all-cause mortality and HF rehospitalization at 2-year follow-up, and each outcome was also separately investigated. Collectively, 382 (16.9%) patients and 37 (12.4%) controls were seropositive for ≥1 AAB (P = 0.045). Seropositivity occurred more frequently only for anti-M2 AABs (P = 0.025). Amongst patients with HF, seropositivity was associated with the presence of comorbidities (renal disease, chronic obstructive pulmonary disease, stroke, and atrial fibrillation) and with medication use. Only anti-ß1 AAB seropositivity was associated with the primary outcome [hazard ratio (95% confidence interval): 1.37 (1.04-1.81), P = 0.024] and HF rehospitalization [1.57 (1.13-2.19), P = 0.010] in univariable analyses but remained associated only with HF rehospitalization after multivariable adjustment for the BIOSTAT-CHF risk model [1.47 (1.05-2.07), P = 0.030]. Principal component analyses showed considerable overlap in B-lymphocyte activity between seropositive and seronegative patients, based on 31 circulating biomarkers related to B-lymphocyte function. CONCLUSIONS: AAB seropositivity was not strongly associated with adverse outcomes in HF and was mostly related to the presence of comorbidities and medication use. Only anti-ß1 AABs were independently associated with HF rehospitalization. The exact clinical value of AABs remains to be elucidated.

Asunto(s)

Autoanticuerpos , Insuficiencia Cardíaca , Humanos , Pronóstico , Receptores Muscarínicos , Receptor Muscarínico M2 , Receptores Adrenérgicos

RESUMEN

BACKGROUND: Oxidative stress may be a key pathophysiological mediator in the development and progression of heart failure (HF). The role of serum-free thiol concentrations, as a marker of systemic oxidative stress, in HF remains largely unknown. OBJECTIVE: The purpose of this study was to investigate associations between serum-free thiol concentrations and disease severity and clinical outcome in patients with new-onset or worsening HF. METHODS: Serum-free thiol concentrations were determined by colorimetric detection in 3802 patients from the BIOlogy Study to TAilored Treatment in Chronic Heart Failure (BIOSTAT-CHF). Associations between free thiol concentrations and clinical characteristics and outcomes, including all-cause mortality, cardiovascular mortality, and a composite of HF hospitalization and all-cause mortality during a 2-years follow-up, were reported. RESULTS: Lower serum-free thiol concentrations were associated with more advanced HF, as indicated by worse NYHA class, higher plasma NT-proBNP (P < 0.001 for both) and with higher rates of all-cause mortality (hazard ratio (HR) per standard deviation (SD) decrease in free thiols: 1.253, 95% confidence interval (CI): 1.171-1.341, P < 0.001), cardiovascular mortality (HR per SD: 1.182, 95% CI: 1.086-1.288, P < 0.001), and the composite outcome (HR per SD: 1.058, 95% CI: 1.001-1.118, P = 0.046). CONCLUSIONS: In patients with new-onset or worsening HF, a lower serum-free thiol concentration, indicative of higher oxidative stress, is associated with increased HF severity and poorer prognosis. Our results do not prove causality, but our findings may be used as rationale for future (mechanistic) studies on serum-free thiol modulation in heart failure. Associations of serum-free thiol concentrations with heart failure severity and outcomes.

Asunto(s)

Insuficiencia Cardíaca , Humanos , Enfermedad Crónica , Gravedad del Paciente , Estrés Oxidativo , Compuestos de Sulfhidrilo/uso terapéutico , Pronóstico , Volumen Sistólico/fisiología

RESUMEN

BACKGROUND: Serum selenium levels have been associated with the incidence of heart failure (HF) and signs of the metabolic syndrome. In addition, notable differences have been reported between males and females in food intake and micronutrient metabolism, possibly explaining different health outcomes. OBJECTIVE: Our objective was to elucidate sex-specific, cross-sectional phenotypic differences in the association of serum selenium concentrations with parameters of metabolic syndrome and HF. METHODS: We investigated data from individuals from a community-based cohort (PREVEND; N = 4288) and heart failure cohort (BIOSTAT-CHF; N = 1994). In both populations, cross-sectional analyses were performed for potential interaction (p < 0.1) between sex and serum selenium with overlapping signs and clinical parameters of the metabolic syndrome and HF. RESULTS: Baseline selenium levels of the total cohort were similar between PREVEND (85.7 µg/L) and BIOSTAT-CHF (89.1 µg/L). Females with lower selenium levels had a higher BMI and increased prevalence of diabetes than females with higher selenium, in both PREVEND (pinteraction < 0.001; pinteraction = 0.040, resp.) and BIOSTAT-CHF (pinteraction = 0.021; pinteraction = 0.024, resp.), while opposite associations were observed for males. Additionally, in females, but not in males, lower selenium was associated with a higher prevalence of myocardial infarction (MI) in PREVEND (pinteraction = 0.021) and BIOSTAT-CHF (pinteraction = 0.084). CONCLUSION: Lower selenium was associated with a higher BMI and increased prevalence of diabetes in females, opposite to males, and was also associated with more MI in females. Interventional studies are needed to validate this observation.

Asunto(s)

Insuficiencia Cardíaca , Síndrome Metabólico , Infarto del Miocardio , Selenio , Masculino , Femenino , Humanos , Síndrome Metabólico/diagnóstico , Síndrome Metabólico/epidemiología , Síndrome Metabólico/complicaciones , Caracteres Sexuales , Prevalencia , Estudios Transversales , Infarto del Miocardio/complicaciones

RESUMEN

AIM: The comorbidities that collectively define metabolic syndrome are common in patients with heart failure. However, the role of metabolic syndrome in the pathophysiology of heart failure is not well understood. We therefore investigated the clinical and biomarker correlates of metabolic syndrome in patients with heart failure. METHODS AND RESULTS: In 1103 patients with heart failure, we compared the biomarker expression using a panel of 363 biomarkers among patients with (n = 468 [42%]) and without (n = 635 [58%]) metabolic syndrome. Subsequently, a pathway overrepresentation analysis was performed to identify key biological pathways. Findings were validated in an independent cohort of 1433 patients with heart failure of whom 615 (43%) had metabolic syndrome. Metabolic syndrome was defined as the presence of three or more of five criteria, including central obesity, elevated serum triglycerides, reduced high-density lipoprotein cholesterol, insulin resistance and hypertension. The most significantly elevated biomarkers in patients with metabolic syndrome were leptin (log2 fold change 0.92, p = 5.85 × 10-21 ), fatty acid-binding protein 4 (log2 fold change 0.61, p = 1.21 × 10-11 ), interleukin-1 receptor antagonist (log2 fold change 0.47, p = 1.95 × 10-13 ), tumour necrosis factor receptor superfamily member 11a (log2 fold change 0.35, p = 4.16 × 10-9 ), and proto-oncogene tyrosine-protein kinase receptor Ret (log2 fold change 0.31, p = 4.87 × 10-9 ). Network analysis identified 10 pathways in the index cohort and 6 in the validation cohort, all related to inflammation. The primary overlapping pathway in both the index and validation cohorts was up-regulation of the natural killer cell-mediated cytotoxicity pathway. CONCLUSION: Metabolic syndrome is highly prevalent in heart failure and is associated with biomarkers and pathways relating to obesity, lipid metabolism and immune responses underlying chronic inflammation.

Asunto(s)

Insuficiencia Cardíaca , Resistencia a la Insulina , Síndrome Metabólico , Humanos , Obesidad , Biomarcadores , Inflamación , Enfermedad Crónica