Basal oxidation of conserved cysteines modulates cardiac titin stiffness and dynamics.

Titin, as the main protein responsible for the passive stiffness of the sarcomere, plays a key role in diastolic function and is a determinant factor in the etiology of heart disease. Titin stiffness depends on unfolding and folding transitions of immunoglobulin-like (Ig) domains of the I-band, and recent studies have shown that oxidative modifications of cryptic cysteines belonging to these Ig domains modulate their mechanical properties in vitro. However, the relevance of this mode of titin mechanical modulation in vivo remains largely unknown. Here, we describe the high evolutionary conservation of titin mechanical cysteines and show that they are remarkably oxidized in murine cardiac tissue. Mass spectrometry analyses indicate a similar landscape of basal oxidation in murine and human myocardium. Monte Carlo simulations illustrate how disulfides and S-thiolations on these cysteines increase the dynamics of the protein at physiological forces, while enabling load- and isoform-dependent regulation of titin stiffness. Our results demonstrate the role of conserved cysteines in the modulation of titin mechanical properties in vivo and point to potential redox-based pathomechanisms in heart disease.

A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues.

Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we specifically sever titin by digestion with TEV protease, and find that the response of muscle fibers to length changes requires mechanical transduction through titin's intact polypeptide chain. In addition, HaloTag-based covalent tethering enables examination of titin dynamics under force using magnetic tweezers. At pulling forces < 10 pN, titin domains are recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Insertion of the HaloTag-TEV cassette in mechanical proteins opens opportunities to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction.

Nutrients restriction upregulates adiponectin in epicardial or subcutaneous adipose tissue: impact in de novo heart failure patients.

Background: Hyperadiponectinemia is an indicator of worse outcomes in advanced heart failure (HF), its role in de novo HF is less clear. Objective: Because this protein is a hormone with starvation properties, we wanted to know its association with nutritional state and its regulator factors in de novo HF. Methods: Adiponectin circulating levels were determined by ELISA at discharge in patients admitted for de novo HF (n=74). Nutritional status was determined by CONUT score. Univariate and multivariate Cox regression analyses were employed to calculate the estimated hazard ratio (HR) with 95% confidence interval (CI) for death or all-cause readmission. Stromal vascular cells (SVC) of EAT and subcutaneous adipose tissue (SAT) from patients (n=5) underwent heart surgery were induced to adipogenesis for 18 days. Then, cells were cultured with complete or starved medium for 8 hours. At the end, adiponectin expression levels were analysed by real time polymerase chain reaction. Results: Patients were grouped regarding nutritional status. There was a strong association between high adiponectin levels and failing nutritional status. Those patients with worse nutritional state had the highest adiponectin and proBNP levels at discharge (p<0.01). Both proteins were slightly correlated (p<0.05). However, only high adiponectin levels were independently associated with death or all-cause readmission. Nutrients starvation upregulated adiponectin expression levels in adipogenesis-induced SVC from EAT or SAT. Conclusions: Worse nutritional state in de novo HF patients is associated with higher adiponectin plasma levels. Their levels were upregulated in adipose cells after being nutrients-starved. These results may help us to understand the adiponectin paradox in HF.

Evolution and bad prognostic value of advanced glycation end products after acute heart failure: relation with body composition.

AIM: The role of advanced glycation end products (AGEs) and their soluble receptor (sRAGE) on the progression and prognosis of acute heart failure (HF) was analysed in relation with metabolic parameters as body composition and nutritional status. METHODS: A hundred and fifty consecutive patients were included in a prospective clinical study during hospitalization by acute HF. Detailed medical history, physical examination, electrocardiogram, echocardiogram and vein peripheral blood were taken for all patients. During the follow-up period [297 days (88-422 days)] blood samples for biochemical measurements were obtained 1 and 6 months after the inclusion. Dual-energy X-ray absorptiometry analyses were performed 1 week after discharge. RESULTS: AGEs and sRAGE levels continuously increased, up to 6 months, after acute HF, but AGEs increase was mainly observed in those patients with incident HF. Both AGEs and sRAGE levels were related with bad renal function and clinical malnutrition (CONUT score) and they were negatively related with body mass index or percentage of body fat. AGEs levels (≥40 a.u.) 1 month after discharge and basal sRAGE levels (>1000 pg/mL) were related with worse prognosis in terms of patient death and HF readmission (Log-rank <0.05 in Kaplan-Meier survival test), independently of age, gender, body mass index and other risk factors. Regression models also corroborated this finding. CONCLUSIONS: AGEs and sRAGE are bad prognostic biomarkers for HF and useful markers of HF progression. Since their levels seem to be related with clinical malnutrition and body composition these parameters could serve to modulate them.

Omentin treatment of epicardial fat improves its anti-inflammatory activity and paracrine benefit on smooth muscle cells.

OBJECTIVE: Epicardial adipose tissue (EAT) in coronary artery disease is insulin resistant and has a proinflammatory profile. This study examined the regulation of EAT by exogenous omentin and its consequence on vascular cells. METHODS: Stromal vascular cells (SC) of EAT and subcutaneous adipose tissue (SAT) from patients who underwent heart surgery were cultured and exposed to adipogenic factors with or without omentin. Proinflammatory cytokine regulation by omentin was analyzed in SC and mature adipocytes. Glucose uptake by EAT and SAT explants was determined after insulin, omentin, or combined treatment. Human vascular cells were exposed to secretomes of SC, with and without omentin treatment. Migration of smooth muscle cells and expression of adhesion molecules were determined by wound healing or real-time polymerase chain reaction, respectively. RESULTS: Omentin treatment raised adipogenesis-induced adiponectin levels on SC of EAT and reduced TNF-α expression levels (0.58 ± 0.14-fold change; P = 0.034) in mature adipocytes. Omentin improved the insulin activity of EAT and SAT explants from cardiovascular disease patients. Finally, secretomes of SC under omentin treatment reduced the migration of smooth muscle cells. CONCLUSIONS: Exogenous omentin might support a cardioprotective role through its effect on EAT regarding glucose uptake, anti-inflammatory response, and its paracrine role on smooth muscle cells.

Orosomucoid as prognosis factor associated with inflammation in acute or nutritional status in chronic heart failure.

BACKGROUND: Inflammation and nutritional state are involved in the pathogenesis of heart failure (HF). OBJECTIVE: To study the contribution of alpha-1-acid-glycoprotein (AGP) to these factors and its prognostic value in acute (AHF) or chronic HF (CHF). METHODS: The observational study has included 147 patients (mean age 70years, 62% men) admitted to a cardiology department for HF and followed-up for an average 326.6±140.8days. Blood AGP values were measured by Enzyme-Linked ImmunoSorbent Assay. Monocytes subsets were determined with CD14 and CD16 antibodies by flow cytometry and body composition was measured by dual-energy X-ray absorptiometry. The regulation of tumor necrosis factor (TNF-α) and leptin by AGP in epicardial adipose tissue (EAT) were analyzed by real time polymerase chain reaction. RESULTS: High AGP, that was associated with CD14+CD16+ monocytes, and proBNP levels at the discharge were indicators of rehospitalization for HF in AHF patients. However, low AGP levels determined a worse nutritional state in CHF patients. The leptin levels were downregulated by high AGP concentration in epicardial fat. CONCLUSION: AGP is a dual indicator in HF because high levels are predictors of adverse outcomes in AHF but low levels are related to the worse nutritional status in CHF. The regulation of leptin by AGP in epicardial fat might suggest a new pathway as protective mechanism in CHF.

Non classical Monocytes Levels, Increased by Subcutaneous Fat-Secretome, Are Associated with Less Rehospitalization after Heart Failure Admission.

Differential monocyte subsets are increased in obesity and heart failure (HF). We studied their role as predictors of rehospitalization for HF and their regulation by adipose tissue. Monocyte subsets and body fat composition were determined from 136 patients at the discharge after HF admission. Regulation of monocytes by SAT secretomes from obese/non-obese patients with HF was studied in a cell culture method. Proteomic analysis of secretome SAT was performed by LC-MALDI TOF/TOF. High CD14-CD16+ monocyte levels indicated less rehospitalization for HF (p = 0.018). SAT secretomes from obese patients increased the CD14-CD16+monocytes (11.8 ± 5.3 vs 3.9 ± 2.6%; p < 0.01). Differential proteins were determined between obese and non-obese patients with HF. High levels of CD14-CD16+ monocytes are associated with less rehospitalization for HF. This phenotype is upregulated by SAT secretome from obese patients with HF. This mechanism might help us to understand the obesity paradox in HF.

Sea cucumbers with an anti-inflammatory effect on endothelial cells and subcutaneous but not on epicardial adipose tissue.

OBJECTIVE: epicardial adipose tissue (EAT) from patients with coronary artery disease (CAD) contains higher levels of inflammatory proteins and lower adiponectin levels than subcutaneous adipose tissue (SAT), enhancing the progression of atherosclerosis. Since products from sea cucumber have anti-inflammatory properties, we investigated its effect on EAT, SAT and endothelial cells. METHODS: stromal cells or explants from EAT and SAT were obtained from patients with cardiovascular disease. Extracts were obtained after hydrolysis by food-grade enzymes at different times. Proteins were identified by LC-MALDI mass spectrometry. Adipogenesis and adiponectin induction were determined on stromal cells in the presence/absence of extracts. The bioavailability of the extracts was tested on a Caco-2 cell culture model in vitro. The bioavailable fraction was probed on endothelial cells and EAT or SAT explants. Vascular cell adhesion protein (VCAM-1), intercellular adhesion molecule (ICAM-1), IL-6 and adiponectin were determined by real time polymerase chain reaction (RT-PCR). RESULTS: our results showed that H. forskali and P. tremulus extracts contained compounds with anti-oxidant and anti-inflammatory properties. The bioavailable fraction of P. tremulus reduced VCAM-1 (p < 0.01) and IL-6 (p < 0.05) expression levels in endothelial cells while bioavailable compounds from H. forskali decreased ICAM-1 expression in SAT (p < 0.05). No effect was observed on EAT. CONCLUSION: these results suggest that sea cucumber extracts might be used for the prevention of endothelial cells and SAT inflammation.

Glucose and Inflammatory Cells Decrease Adiponectin in Epicardial Adipose Tissue Cells: Paracrine Consequences on Vascular Endothelium.

Epicardial adipose tissue (EAT) is a source of energy for heart that expresses the insulin-sensitizer, anti-inflammatory and anti-atherogenic protein, adiponectin. But, in coronary artery disease, adiponectin production declines. Our objective was to determine its regulation by glucose and inflammation in stromal cells from EAT and subcutaneous adipose tissue (SAT) and its paracrine effect on endothelial cells. Stromal cells of EAT and SAT were obtained from patients who underwent cardiac surgery. Adipogenesis was induced at 117, 200, or 295 mg/dl glucose, with or without macrophage-conditioned medium (MCM). Expression of adiponectin, GLUT-4 and the insulin receptor was analyzed by real-time PCR. The paracrine effect of stromal cells was determined in co-cultures with endothelial cells, by exposing them to high glucose and/or MCM, and, additionally, to leukocyte-conditioned medium from patients with myocardial infarction. The endothelial response was determined by analyzing vascular adhesion molecule expression. Our results showed a U-shaped dose-response curve of glucose on adiponectin in EAT, but not in SAT stromal cells. Conversely, MCM reduced the adipogenesis-induced adiponectin expression of EAT stromal cells. The presence of EAT stromal increased the inflammatory molecules of endothelial cells. This deleterious effect was emphasized in the presence of inflammatory cell-conditioned medium from patients with myocardial infarction. Thus, high glucose and inflammatory cells reduced adipogenesis-induced adiponectin expression of EAT stromal cells, which induced an inflammatory paracrine process in endothelial cells. This inflammatory effect was lower in presence of mature adipocytes, producers of adiponectin. These results contribute to understanding the role of EAT dysfunction on coronary atherosclerosis progression.

Differential behaviour of epicardial adipose tissue-secretomes with high and low orosomucoid levels from patients with cardiovascular disease in H9C2 cells.

Epicardial adipose tissue releases orosomucoid (ORM), an acute phase protein with multiple modulatory and protective properties. We aimed to identify the effect of EAT-supernatants according to their ORM levels on H9C2 cells. H9C2 were cultured with EAT-secretomes or ORM protein itself on a Real-Time Cell Analyser. Secretome proteins identification was performed by LC-mass spectrometry according to their ORM levels. Two of them were validated by ELISA in EAT-supernatants from 42 patients. ORM effect on H9C2 and neonatal rat cardiomyocytes apoptosis under hypoxia with or without fatty acid treatment was determined by Annexin-V flow cytometry measurement. Caspase-3 expression levels were determined by western blot in H9C2. Our results showed a differential effect of EAT-secretomes according their ORM levels. Although additional secreted proteins can contribute to their beneficial effects, ORM reduced hypoxia-induced apoptosis through caspase-3 inhibition. Our data showed the cardioprotective role of ORM and suggest that its quantification on EAT secretomes might help us to find new secreted factors with a cardioprotective role.

Orosomucoid secretion levels by epicardial adipose tissue as possible indicator of endothelial dysfunction in diabetes mellitus or inflammation in coronary artery disease.

OBJECTIVE: Type 2 diabetes mellitus (T2DM) is associated with fat and autonomic system dysfunction. Epicardial adipose tissue (EAT) plays an endocrine role over the heart. Since orosomucoid (ORM) has local actions around the coronaries, our aim was to assess the relationship between its secretion profile by EAT and its catecholaminergic regulation in patients with T2DM and coronary artery disease (CAD). METHODS: We obtained EAT, subcutaneous adipose tissue (SAT) and plasma from 55 patients undergoing cardiac surgery. Fat explants were stimulated with isoproterenol (ISO) 1 µM for 6 h. After, the fat explants released-ORM and plasma levels were analyzed by ELISA. mRNA or protein expression was analyzed by real time PCR or western blot, respectively. The effects of ORM on endothelial cells were analyzed by impedance and wound healing assays. RESULTS: We observed that EAT-released ORM levels were higher than SAT (328 ± 185 vs 58 ± 45 ng/mL; p < 0.001). Interestingly, EAT secretion was lower in patients with than those without T2DM (260 ± 141 vs 370 ± 194 ng/mL; p < 0.05) and this difference was enhanced after ISO stimulation (p < 0.01). However, plasma levels (412 ± 119 vs 594 ± 207 µg/mL) and EAT-released ORM levels were higher in patients with than those without CAD (384 ± 195 vs 279 ± 159 ng/mL; p < 0.05). ISO stimulation, also reduced the EAT released-ORM levels in patients with CAD. On human endothelial cells, ORM induced an increase of healing and proliferation in a dose-dependent manner. CONCLUSION: EAT-released ORM levels in patients with T2DM or CAD and its regulation by catecholamines might be the mirror of local endothelium dysfunction or inflammatory process in different cardiovascular disorders.

Differential association of S100A9, an inflammatory marker, and p53, a cell cycle marker, expression with epicardial adipocyte size in patients with cardiovascular disease.

S100A9 (calgranulin B) has inflammatory and oxidative stress properties and was found to be associated with atherosclerosis and obesity. One of the proteins that can regulate S100A9 transcription is p53, which is involved in cell cycle, apoptosis and adipogenesis. Thus, it triggers adipocyte enlargement and finally obesity. Because epicardial adipose tissue (EAT) volume and thickness is related to coronary artery disease (CAD), we studied the gene expression of this pathway in patients with cardiovascular disease and its association with obesity. Adipocytes and stromal cells from EAT and subcutaneous adipose tissue (SAT) from 48 patients who underwent coronary artery bypass graft and/or valve replacement were obtained after collagenase digestion and differential centrifugation. The expression levels of the involved genes on adipogenesis and cell cycle like fatty acid-binding protein (FABP) 4, retinol-binding protein (RBP)4, p53 and S100A9 were determined by real-time polymerase chain reaction (PCR). Adipocyte diameter was measured by optical microscopy. We found that epicardial adipocytes expressed significantly lower levels of adipogenic genes (FABP4 and RBP4) and cell cycle-related genes (S100A9 and p53) than subcutaneous adipocytes. However, in obese patients, upregulation of adipogenic and cell cycle-related genes in subcutaneous and epicardial adipocytes, respectively, was observed. The enlargement of adipocyte size was related to FABP4, S100A9 and p53 expression levels in stromal cells. But only the p53 association was maintained in epicardial stromal cells from obese patients (p=0.003). The expression of p53, but not S100A9, in epicardial stromal cells is related to adipocyte enlargement in obese patients with cardiovascular disease. These findings suggest new mechanisms for understanding the relationship between epicardial fat thickness, obesity and cardiovascular disease.

Impaired adipogenesis and insulin resistance in epicardial fat-mesenchymal cells from patients with cardiovascular disease.

The thickness of epicardial adipose tissue (EAT), which is an inflammatory source for coronary artery disease (CAD), correlates with insulin resistance. One trigger factor is impaired adipogenesis. Here, our aim was to clarify the underlying mechanisms of insulin resistance on EAT-mesenchymal cells (MC). EAT and subcutaneous adipose tissue (SAT) were collected from 19 patients who were undergoing heart surgery. Their dedifferentiated adipocytes (DAs) and/or MCs were cultured. After the induction of adipogenesis or stimulation with insulin, the expression of adipokines was analyzed using real-time polymerase chain reaction (PCR). Colorimetric assays were performed to measure glucose levels and proliferation rate. Proteins modifications were detected via the proteomic approach and Western blot. Our results showed lower adipogenic ability in EAT-MCs than in SAT-MCs. Maximum adiponectin levels were reached within 28-35 days of exposure to adipogenic inducers. Moreover, the adipogenesis profile in EAT-MCs was dependent on the patients' clinical characteristics. The low adipogenic ability of EAT-MCs might be associated with an insulin-resistant state because chronic insulin treatment reduced the inflammatory cytokine expression levels, improved the glucose consumption, and increased the post-translational modifications (PTMs) of the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1). We found lower adipogenic ability in EAT-MCs than in SAT-MCs. This lower ability level was dependent on gender and the presence of diabetes, obesity, and CAD. Low adipogenesis ability and insulin resistance in EAT-MCs might shed light on the association between EAT dysfunction and cardiovascular disease.

Differential behavior between S100A9 and adiponectin in coronary artery disease. Plasma or epicardial fat.

AIMS: S100A9 is a new inflammatory marker associated with obesity and cardiovascular disease. Because epicardial adipose tissue (EAT) is an inflammatory source in coronary artery disease (CAD), our aim was to evaluate the S100A9 levels in plasma and EAT and its association with CAD. MAIN METHODS: Blood, EAT and/or subcutaneous adipose tissue (SAT) biopsies were obtained from 89 patients undergoing elective cardiac surgery. Plasma S100A9 and adiponectin were analyzed by enzyme-linked immunosorbent assay (ELISA) and mRNA expression in both fat pads and were measured by real-time polymerase chain reaction (PCR). KEY FINDINGS: Our results have shown higher levels of plasma S100A9 in patients with CAD than those without (29 [10-50] vs. 17 [3-28] ng/mL; p=0.007). They were dependent on the number of injured-coronaries (p=0.002) with tendency toward negative association with plasma adiponectin (p=0.139). Although EAT expressed higher levels than SAT and their levels were higher in CAD patients, this last difference did not reach statistical significance. However, there was a positive correlation between neutrophils and EAT S100A9 expression (p=0.007) that may reveal an increase of neutrophil filtration on this fat pad. SIGNIFICANCE: Plasma S100A9 levels are increased in chronic CAD. The absence of differences regarding EAT S100A9 expression levels indicates a differential inflammatory process between fat tissues and blood in CAD process.

Adiponectin and p53 mRNA in epicardial and subcutaneous fat from heart failure patients.

OBJECTIVE: Heart failure (HF) is associated with a pro-inflammatory state in epicardial fat, but the involved mechanisms are not entirely clear. The aim of our study was to assess the relationship between p53 and adiponectin mRNA in epicardial adipose tissue (EAT) and subcutaneous adipose tissue (SAT) in patients with heart failure and its sympathetic regulation. METHODS: Epicardial adipose tissue and SAT samples were obtained from 63 patients undergoing elective cardiac surgery. EAT and SAT explants culture from seven patients were stimulated with isoprenaline 0.1 or 1 uM for 6 h. p53 and adiponectin mRNA expression was measured in frozen biopsies or explants culture from both fat pads by real-time polymerase chain reaction (PCR). RESULTS: We observed that EAT expressed more p53 mRNA than SAT (1.73 ± 0.07 vs. 1.69 ± 0.04, P < 0.001) and its levels were higher in HF patients (1.75 ± 0.07 vs. 1.70 ± 0.04, P < 0.01 in EAT and 1.70 ± 0.04 vs. 1.67 ± 0.04, P < 0.05 in SAT). Moreover, p53 mRNA expression was negatively correlated with adiponectin in EAT. After analysing the p53 mRNA regulation by isoprenaline, we observed that only EAT p53 expression increased after adrenergic stimulation (1.63 ± 0.01 vs. 1.66 ± 0.02; P = 0.024). CONCLUSIONS: p53 mRNA expression levels, inversely correlated with adiponectin, increase in EAT of HF patients and can be regulated by sympathetic activation pathway. Our findings can help to explain the deleterious effect of sympathetic activation in HF.