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Sulfonylureas (SUs) are a class of antidiabetic drugs widely used in the management of diabetes mellitus type 2. They promote insulin secretion by inhibiting the ATP-sensitive potassium channel in pancreatic β-cells. Recently, the exchange protein directly activated by cAMP (Epac) was identified as a new class of target proteins of SUs that might contribute to their antidiabetic effect, through the activation of the Ras-like guanosine triphosphatase Rap1, which has been controversially discussed. We used human embryonic kidney (HEK) 293 cells expressing genetic constructs of various Förster resonance energy transfer (FRET)-based biosensors containing different versions of Epac1 and Epac2 isoforms, alone or fused to different phosphodiesterases (PDEs), to monitor SU-induced conformational changes in Epac or direct PDE inhibition in real time. We show that SUs can both induce conformational changes in the Epac2 protein but not in Epac1, and directly inhibit the PDE3 and PDE4 families, thereby increasing cAMP levels in the direct vicinity of these PDEs. Furthermore, we demonstrate that the binding site of SUs in Epac2 is distinct from that of cAMP and is located between the amino acids E443 and E460. Using biochemical assays, we could also show that tolbutamide can inhibit PDE activity through an allosteric mechanism. Therefore, the cAMP-elevating capacity due to allosteric PDE inhibition in addition to direct Epac activation may contribute to the therapeutic effects of SU drugs.

Risk stratification among patients with coronary artery disease (CAD) is of considerable interest to potentially guide secondary preventive therapies. Cardiac troponins as well as C-reactive protein (hsCRP) and natriuretic peptides have emerged as biomarkers for risk stratification. The question remains if one of these biomarkers is superior in predicting adverse outcomes. Thus, we perform a head-to-head comparison between high-sensitivity troponin I (hsTnI), hsCRP, and N-terminal pro-brain natriuretic peptide (NT-proBNP) in patients with CAD. Plasma levels were measured in a cohort of 2193 patients with documented CAD. The main outcome measures were cardiovascular (CV) death and/or nonfatal myocardial infarction (MI). During a median follow-up of 3.8 years, all three biomarkers were associated with cardiovascular death and/or MI. After adjustments for conventional cardiovascular risk factors, the hazard ratio (HR) per standard deviation (SD) for the prediction of CV death and/or nonfatal MI was 1.39 [95% CI: 1.24−1.57, p < 0.001] for hsTnI, 1.41 [95% CI: 1.24−1.60, p < 0.001] for hsCRP, and 1.64 [95% CI: 1.39−1.92, p < 0.001] for NT-proBNP. However, upon further adjustments for the other two biomarkers, only NT-proBNP was still associated with the combined endpoint with an HR of 1.47 [95% CI: 1.19−1.82, p < 0.001]. Conclusively, NT-proBNP is reliably linked to CV death and MI in patients with CAD and provides incremental value beyond hsCRP and hsTnI.

High-sensitivity troponin has proven to be a promising biomarker for the prediction of future adverse cardiovascular events. We aimed to assess the prognostic value of high-sensitivity troponin I (hs-TnI) on admission in patients with suspected acute myocardial infarction (AMI) analyzed by a novel (Singulex Clarity cTnI) and established hs-TnI assay (ARCHITECT STAT hs-TnI, Abbott). Hs-TnI was measured in a total of 2332 patients from two prospective cohort studies presenting to the emergency department with suspected AMI. The prognostic impact for overall and cardiovascular mortality of both hs-TnI assays was assessed in the total patient cohort as well as in the subgroups of patients with AMI (n = 518) and without AMI (non-AMI) (n = 1814). Patients presenting with highest hs-TnI levels showed higher overall and cardiovascular mortality rates compared to those with lower troponin levels, irrespective of the assay used. Both hs-TnI assays indicated association with overall mortality according to adjusted hazard ratio (HR) among the entire study population (HR for Singulex assay: 1.16 (95% CI 1.08−1.24) and HR for Abbott assay: 1.17 (95% CI 1.09−1.25)). This finding was particularly pronounced in non-AMI patients, whereas no association between hs-TnI and overall mortality was found in AMI patients for either assay. In non-AMI patients, both assays equally improved risk prediction for cardiovascular mortality beyond conventional cardiovascular risk factors. Hs-TnI is independently predictive for adverse outcomes in patients with suspected AMI, especially in the subset of patients without confirmed AMI. There was no difference between the established and the novel assay in the prediction of mortality.

(1) Background: Telomeres are repetitive DNA sequences located at the extremities of chromosomes that maintain genetic stability. Telomere biology is relevant to several human disorders and diseases, specifically cardiovascular disease. To better understand the link between cardiovascular disease and telomere length, we studied the effect of relative telomere length (RTL) on cardiovascular risk factors in a large population-based sample. (2) Methods: RTL was measured by a real-time quantitative polymerase chain reaction in subjects of the population-based Gutenberg Health Study (n = 4944). We then performed an association study of RTL with known cardiovascular risk factors of smoking status as well as systolic and diastolic blood pressure, body mass index (BMI), LDL cholesterol, HDL cholesterol, and triglycerides. (3) Results: A significant correlation was shown for RTL, with age as a quality control in our study (effect = −0.004, p = 3.2 × 10−47). Analysis of the relation between RTL and cardiovascular risk factors showed a significant association of RTL in patients who were current smokers (effect = −0.016, p = 0.048). No significant associations with RTL were seen for cardiovascular risk factors of LDL cholesterol (p = 0.127), HDL cholesterol (p = 0.713), triglycerides (p = 0.359), smoking (p = 0.328), diastolic blood pressure (p = 0.615), systolic blood pressure (p = 0.949), or BMI (p = 0.903). In a subsequent analysis, we calculated the tertiles of RTL. No significant difference across RTL tertiles was detectable for BMI, blood pressure, lipid levels, or smoking status. Finally, we studied the association of RTL and cardiovascular risk factors stratified by tertiles of age. We found a significant association of RTL and LDL cholesterol in the oldest tertile of age (effect = 0.0004, p = 0.006). (4) Conclusions: We determined the association of relative telomere length and cardiovascular risk factors in a population setting. An association of telomere length with age, current smoking status, as well as with LDL cholesterol in the oldest tertile of age was found, whereas no associations were observed between telomere length and triglycerides, HDL cholesterol, blood pressure, or BMI.

The soluble urokinase-type plasminogen activator receptor (suPAR) is a new marker for immune activation and inflammation and may provide diagnostic value on top of established biomarkers in patients with suspected acute myocardial infarction (AMI). Here, we evaluate the diagnostic potential of suPAR levels on top of high-sensitivity troponin I (hs-TnI) in a cohort of patients with suspected AMI. A total of 1220 patients presenting to the emergency department with suspected AMI were included, of whom 245 were diagnosed with AMI. Median suPAR levels at admission were elevated in subjects with AMI compared to non-AMI (3.8 ng/mL vs 3.3 ng/mL, p = 0.001). In C-statistics, the area under the curve (AUC) regarding the diagnosis of AMI was low (0.57 at an optimized cut-off of 3.7 ng/mL). Moreover, baseline suPAR levels on top of troponin values at admission and hour 1 reduced the number of patients who were correctly ruled-out as non-AMI, and who were correctly ruled-in as AMI. Our study shows that circulating levels of suPAR on top of high-sensitivity troponin I do not improve the early diagnosis of AMI.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine known to play a major role in inflammatory diseases such as myocardial infarction (MI), where its expression increases. Cardio protective functions of MIF during ischemia have been reported. Recently, the structurally related MIF-2 was identified and similar effects are assumed. We wanted to further investigate the role of MIF and MIF-2 on inflammatory processes during MI. Therefore, we subjected mice to experimentally induced MI by coronary occlusion for one and five days. During the acute phase of MI, the gene expression of Mif was upregulated in the infarct zone, whereas Mif-2 was downregulated, suggesting a minor role of MIF-2. Simulating ischemic conditions or mechanical stress in vitro, we demonstrated that Mif expression was induced in resident cardiac cells. To investigate possible auto /paracrine effects, cardiomyocytes and cardiac fibroblasts were individually treated with recombinant murine MIF, which in turn induced Mif expression and the expression of pro-inflammatory genes in cardiac fibroblasts. Cardiomyocytes did not respond to recombinant MIF with pro-inflammatory gene expression. While MIF stimulation alone did not change the expression of pro-fibrotic genes in cardiac fibroblasts, ischemia reduced their expression. Mimicking the increased MIF levels during MI, we exposed cardiac fibroblasts to simulated ischemia in the presence of MIF, which led to further reduced expression of pro-fibrotic genes. The presented data show that MIF was expressed by resident cardiac cells during MI. In vitro, Mif expression was induced by different external stimuli in cardiomyocytes and cardiac fibroblasts. Addition of recombinant MIF protein increased the expression of pro-inflammatory genes in cardiac fibroblasts including Mif expression itself. Thereby, cardiac fibroblasts may amplify Mif expression during ischemia promoting cardiomyocyte survival.

Homoarginine has come into the focus of interest as a biomarker for cardiovascular disease. Atrial fibrillation (AF) causes a substantial increase in morbidity and mortality. Whether circulating homoarginine is associated with occurrence or persistence of AF and may serve as a new predictive biomarker remains unknown. We measured plasma levels of homoarginine in the population-based Gutenberg health study (3761 patients included, of them 51.7% males), mean age 55.6 ± 10.9 years-old. Associations between homoarginine and intermediate electrocardiographic and echocardiographic phenotypes and manifest AF were examined. Patients with AF (124 patients, of them 73.4% males) had a mean age 64.8 ± 8.6 years-old compared to a mean age of 55.3 ± 10.9 in the population without AF (p-value < 0.001) and showed a less beneficial risk factor profile. The median homoarginine levels in individuals with and without AF were 1.9 μmol/L (interquartile range (IQR) 1.5–2.5) and 2.0 μmol/L (IQR 1.5–2.5), respectively, p = 0.56. In multivariable-adjusted regression analyses homoarginine was not statistically significantly related to electrocardiographic variables. Among echocardiographic variables beta per standard deviation increase was −0.12 (95% confidence interval (CI) −0.23–(−0.02); p = 0.024) for left atrial area and −0.01 (95% CI −0.02–(−0.003); p = 0.013) for E/A ratio. The odds ratio between homoarginine and AF was 0.91 (95% CI 0.70–1.16; p = 0.45). In our large, population-based cross-sectional study, we did not find statistically significant correlations between lower homoarginine levels and occurrence or persistence of AF or most standard electrocardiographic phenotypes, but some moderate inverse associations with echocardiographic left atrial size and E/A. Homoarginine may not represent a strong biomarker to identify individuals at increased risk for AF. Further investigations will be needed to elucidate the role of homoarginine and cardiac function.

Smoking is a major risk factor for cardiovascular diseases and has been implicated in the regulation of the G protein-coupled receptor 15 (GPR15) by affecting CpG methylation. The G protein-coupled receptor 15 is involved in angiogenesis and inflammation. An effect on GPR15 gene regulation has been shown for the CpG site CpG3.98251294. We aimed to analyze the effect of smoking on GPR15 expression and methylation sites spanning the GPR15 locus. DNA methylation of nine GPR15 CpG sites was measured in leukocytes from 1291 population-based individuals using the EpiTYPER. Monocytic GPR15 expression was measured by qPCR at baseline and five-years follow up. GPR15 gene expression was upregulated in smokers (beta (ß) = −2.699, p-value (p) = 1.02 × 10−77) and strongly correlated with smoking exposure (ß = −0.063, p = 2.95 × 10−34). Smoking cessation within five years reduced GPR15 expression about 19% (p = 9.65 × 10−5) with decreasing GPR15 expression over time (ß = 0.031, p = 3.81 × 10−6). Additionally, three novel CpG sites within GPR15 affected by smoking were identified. For CpG3.98251047, DNA methylation increased steadily after smoking cessation (ß = 0.123, p = 1.67 × 10−3) and strongly correlated with changes in GPR15 expression (ß = 0.036, p = 4.86 × 10−5). Three novel GPR15 CpG sites were identified in relation to smoking and GPR15 expression. Our results provide novel insights in the regulation of GPR15, which possibly linked smoking to inflammation and disease progression.

Most studies reporting on the association of circulating testosterone levels with type 2 diabetes in men are of cross-sectional design. Reports on the relevance of altered testosterone levels in women are scarce. Here, we evaluate the role of low serum testosterone levels for incident diabetes in men and women in a population setting of 7706 subjects (3896 females). During a mean follow up time of 13.8 years, 7.8% developed type 2 diabetes. Significant correlations of testosterone with high density lipoprotein (HDL)-cholesterol (R = 0.21, p < 0.001), body-mass-index (R = −0.23, p < 0.001), and waist-to-hip-ratio (R = −0.21, p < 0.001) were found in men. No correlation was found with age in men; in women, the correlation was negligible (R = 0.04, p = 0.012). In men, low testosterone levels predicted high risk of type 2 diabetes, while in women this relationship was opposite. Men with low testosterone levels showed increased risk of future diabetes (hazard ratio (HR) 2.66, 95% confidence interval (CI) 1.91–3.72, p < 0.001 in basic model; HR 1.56 95%, CI 1.10–2.21, p = 0.003). In women, low testosterone levels indicated lower risk with (HR 0.53, 95% CI 0.37–0.77, p = 0.003), while the association lost significance in the fully adjusted model (HR 0.72, 95% CI 0.49–1.05, p = 0.09). Low levels of testosterone predicted future diabetes in men. A borderline opposite association was found in women.

Intrinsic iron release is discussed to have favorable effects in coronary artery disease (CAD). The aim of this study was to evaluate the prognostic relevance of intrinsic iron release in patients with CAD. Intrinsic iron release was based on a definition including hepcidin and soluble transferrin receptor (sTfR). In a cohort of 811 patients with angiographically documented CAD levels of hepcidin and sTfR were measured at baseline. Systemic body iron release was defined as low levels of hepcidin (

Acute myocardial infarction remains a leading cause of morbidity and mortality. While iron deficient heart failure patients are at increased risk of future cardiovascular events and see improvement with intravenous supplementation, the clinical relevance of iron deficiency in acute coronary syndrome remains unclear. We aimed to evaluate the prognostic value of iron deficiency in the acute coronary syndrome (ACS). Levels of ferritin, iron, and transferrin were measured at baseline in 836 patients with ACS. A total of 29.1% was categorized as iron deficient. The prevalence of iron deficiency was clearly higher in women (42.8%), and in patients with anemia (42.5%). During a median follow-up of 4.0 years, 111 subjects (13.3%) experienced non-fatal myocardial infarction (MI) and cardiovascular mortality as combined endpoint. Iron deficiency strongly predicted non-fatal MI and cardiovascular mortality with a hazard ratio (HR) of 1.52 (95% confidence interval (CI) 1.03-2.26; p = 0.037) adjusted for age, sex, hypertension, smoking status, diabetes, hyperlipidemia, body-mass-index (BMI) This association remained significant (HR 1.73 (95% CI 1.07–2.81; p = 0.026)) after an additional adjustment for surrogates of cardiac function and heart failure severity (N-terminal pro B-type natriuretic peptide, NT-proBNP), for the size of myocardial necrosis (troponin), and for anemia (hemoglobin). Survival analyses for cardiovascular mortality and MI provided further evidence for the prognostic relevance of iron deficiency (HR 1.50 (95% CI 1.02–2.20)). Our data showed that iron deficiency is strongly associated with adverse outcome in acute coronary syndrome.

Iron is essential in terms of oxygen utilization and mitochondrial function. The liver-derived peptide hepcidin has been recognized as a key regulator of iron homeostasis. Since iron metabolism is crucially linked to cardiovascular health, and low hepcidin was proposed as potential new marker of iron metabolism, we aimed to evaluate the prognostic value of hepcidin in a large cohort of patients with coronary heart disease (CHD). Serum levels of hepcidin were determined at baseline in patients with angiographically documented CHD. The main outcome measure was non-fatal myocardial infarction (MI) or cardiovascular death. During a median follow-up of 4.1 years, 10.3% experienced an endpoint. In Cox regression analyses for hepcidin the hazard ratio for future cardiovascular death or MI was 1.03 (95% confidence interval (CI) 0.91–1.18, p = 0.63) after adjustment for sex and age. This association virtually did not change after additional adjustment for body mass index (BMI), smoking status, hypertension, diabetes, dyslipidemia, and surrogates of cardiac function (NT-proBNP), size of myocardial necrosis (troponin I), and anemia (hemoglobin). In this study, by far the largest evaluating the predictive value of hepcidin, hepcidin levels were not associated with future MI or cardiovascular death. This implicates a limited, if any, role for hepcidin in secondary cardiovascular risk prediction.