DNA hydroxymethylation controls cardiomyocyte gene expression in development and hypertrophy.

Methylation at 5-cytosine (5-mC) is a fundamental epigenetic DNA modification associated recently with cardiac disease. In contrast, the role of 5-hydroxymethylcytosine (5-hmC)-5-mC's oxidation product-in cardiac biology and disease is unknown. Here we assess the hydroxymethylome in embryonic, neonatal, adult and hypertrophic mouse cardiomyocytes, showing that dynamic modulation of hydroxymethylated DNA is associated with specific transcriptional networks during heart development and failure. DNA hydroxymethylation marks the body of highly expressed genes as well as distal regulatory regions with enhanced activity. Moreover, pathological hypertrophy is characterized by a shift towards a neonatal 5-hmC distribution pattern. We also show that the ten-eleven translocation 2 (TET2) enzyme regulates the expression of key cardiac genes, such as Myh7, through 5-hmC deposition on the gene body and at enhancers. Thus, we provide a genome-wide analysis of 5-hmC in the cardiomyocyte and suggest a role for this epigenetic modification in heart development and disease.

Comparison of the new high sensitive cardiac troponin T with myoglobin, h-FABP and cTnT for early identification of myocardial necrosis in the acute coronary syndrome.

BACKGROUND: We sought to determine the performance of the new high sensitivity cardiac troponin T assay (TnThs) for early diagnosis of myocardial infarction in patients with suspected acute coronary syndrome (ACS) and compare it with the fourth generation cTnT assay, myoglobin and heart-type fatty acid binding protein (h-FABP). METHODS: Ninety-four patients with diagnosis of suspected ACS without ST-segment elevation admitted to our chest pain unit were included. Patients were divided according to time from onset of symptoms to presentation into an early presenter group (<4 h) and a late presenter group (≥4 h). A median of six samples (range 2-8) were available per patient. The diagnostic performance of TnThs was assessed using ROC analysis. Areas under the curve (AUC) of baseline and follow-up results of TnThs, cTnT, myoglobin, and h-FABP were compared using c statistics. RESULTS: The TnThs assay allows an excellent prediction of non-ST-segment elevation myocardial infarction (non-STEMI) at presentation, particularly among late presenters. A follow-up sample improves diagnostic performance in a time-dependent manner. The AUC of TnThs was superior to cTnT at all time points. The performance of TnThs was at least as good as myoglobin and h-FABP at presentation and during follow-up. CONCLUSIONS: A baseline sample of TnThs allows an earlier prediction of non-STEMI than the less sensitive and precise fourth generation cTnT assay. Probably, this excellent performance of TnThs at baseline and follow-up could obviate the need for other early markers of necrosis in future.

High-sensitivity cardiac troponin T for early prediction of evolving non-ST-segment elevation myocardial infarction in patients with suspected acute coronary syndrome and negative troponin results on admission.

BACKGROUND: We sought to determine the diagnostic performance of the new high-sensitivity cardiac troponin T (hs-cTnT) assay for early detection of non-ST-segment myocardial infarction (NSTEMI) in patients with acute coronary syndrome. METHODS: We enrolled patients with retrospectively confirmed unstable angina or NSTEMI and an initially negative cTnT concentration and compared the performance of baseline concentrations and serial changes in concentration within 3 and 6 h. Percentage change criteria included >or=20% delta change and ROC-optimized value. RESULTS: Based on the standard fourth-generation cTnT result of >or=0.03 microg/L, an evolving NSTEMI was diagnosed in 26 patients, and 31 patients were classified as having unstable angina. With the use of the hs-cTnT assay at the 99th-percentile cutoff, the percentage of NSTEMI cases detected increased gradually from 61.5% on presentation to 100% within 6 h, and the overall number of MI diagnoses increased by 34.6% (35 vs 26 cases). A delta change >or=20% or >or=ROC-optimized value of >117% within 3 h or >or=243% within 6 h yielded a specificity of 100% at sensitivities between 69% and 76%. The standard cTnT at the 99th percentile was less sensitive than hs-cTnT for early diagnosis of MI on presentation, and follow-up samples obtained within the initial 3 h demonstrated very low specificity of cTnT compared with hs-cTnT. CONCLUSIONS: The high-sensitivity cTnT assay increases the number of NSTEMI diagnoses and enables earlier detection of evolving NSTEMI. A doubling of the hs-cTnT concentration within 3 h in the presence of a second concentration >or=99th percentile is associated with a positive predictive value of 100% and a negative predictive value of 88%.

Analytical validation of a high-sensitivity cardiac troponin T assay.

BACKGROUND: We report the development of a novel high-sensitivity cardiac troponin T (hs-cTnT) assay, a modification of the Roche fourth-generation cTnT assay, and validation of the analytical performance of this assay. METHODS: Validation included testing of analytical sensitivity, specificity, interferences, and precision. We established the 99th percentile cutoff from healthy reference populations (n = 616). In addition, we studied differences in time to a positive result when using serial measurements of hs-cTnT vs cTnT in patients with a confirmed diagnosis of non-ST elevation myocardial infarction (non-STEMI). RESULTS: The hs-cTnT assay had an analytical range from 3 to 10 000 ng/L. At the 99th percentile value of 13.5 ng/L, the CV was 9% using the Elecsys 2010 analyzer. The assay was specific for cTnT without interferences from human cTnI or cTnC, skeletal muscle TnT, or hemoglobin concentrations up to 1000 mg/L, above which falsely lower values would be expected. When the assay was evaluated clinically, a hs-cTnT higher than the 99th percentile concentration identified a significantly higher number of patients with non-STEMI on presentation (45 vs 20 patients, P = 0.0004) compared with cTnT, and a final diagnosis of non-STEMI was made in 9 additional patients (55 vs 46 patients, P = 0.23) after serial sampling. Time to diagnosis was significantly shorter using hs-cTnT compared with cTnT [mean 71.5 (SD 108.7) min vs 246.9 (82.0) min, respectively; P < 0.01]. CONCLUSIONS: The analytical performance of hs-cTnT complies with the ESC-ACCF-AHA-WHF Global Task Force recommendations for use in the diagnosis of MI.

Serial and single time-point measurements of cardiac troponin T for prediction of clinical outcomes in patients with acute ST-segment elevation myocardial infarction.

BACKGROUND: Cardiac troponins are the preferred biomarkers to predict infarct size in patients (pts) after acute myocardial infarction (AMI). Less information is currently available to verify the prognostic value of such a biomarker surrogate. METHODS: We included 82 pts with acute STEMI and compared all single time point and serial cardiac troponin T (cTnT) values (peak and area-under-the-curve) from admission until day 4 to predict future major adverse cardiac events (MACE). RESULTS: Pts who had suffered any MACE during follow-up had higher cTnT values (median (25th/75th percentiles) on day 4 (3.16 microg/l (2.71/5.20) Vs. 2.1 microg/l (1.19/3.96), P=0.0304), and higher peak cTnT values (5.11 microg/l (3.31/9.47) Vs. 2.92 microg/l (1.81/5.63), P=0.0234). The likelihood to develop a composite of MACE was twofold higher in the intermediate cTnT tertile (1.66-3.04 microg/l, n=23), and in the upper cTnT tertile (3.35-20.68 microg/l, n=23) for cTnT on day 4. For cTnT peak the risk was 1.7-fold higher in the intermediate cTnT peak tertile (2.55-5.01 microg/l, n=28) and 2.4-fold in the upper cTnT peak tertile (5.11-18.93 microg/l, n=27). The optimal ROC cutoff for cTnT to predict the composite of MACE was 2.69 microg/l measured on day 4 and 2.85 microg/l for the cTnT peak. CONCLUSIONS: A single measurement of cTnT after STEMI is an independent predictor for MACE, performs as effective as serial cTnT sampling and may be useful to assess future events.

Platelet reactivity and clopidogrel resistance are associated with the H2 haplotype of the P2Y12-ADP receptor gene.

BACKGROUND: Platelet hyperreactivity was reported in clopidogrel-naiotave carriers of the H2 haplotype of the P2Y(12) platelet ADP receptor. Here, we studied the influence of this genetic variant on clopidogrel responsiveness. METHODS: ADP-mediated (5 micromol/L) platelet aggregation was determined by impedance (Omega) aggregometry in 43 clopidogrel-naïve blood donors and 557 patients treated with aspirin and clopidogrel after percutaneous coronary stent implantation. A cut-off of 5 Omega was used to classify the aggregation response. Haplotype tagging single nucleotide polymorphism G52T was genotyped using a TaqMan assay. RESULTS: The number of H2 alleles correlated with aggregation in clopidogrel-naïve subjects in healthy subjects (p=0.041): impedance results were 8.4+/-3.6, 10.5+/-1.6 and 12.5+/-2.1 Omega in carriers of the H1/H1 (n=30), H1/H2 (n=11) and H2/H2 (n=2) haplotypes, respectively. 87.1% (n=485) and 12.9% (n=72) of clopidogrel treated patients were responders and nonresponders, respectively. Women were more likely to be nonresponders (O.R. 3.90 [95% CI 2.34-6.50]). Carriers of a H2/H2 haplotype (n=14) exhibited stronger aggregation than patients with at least one H1 allele (6.3+/-7.5 vs. 1.8+/-3.3 Omega, p=0.0212) and were more frequently nonresponders (p=0.004). Consequently, the H2/H2 haplotype was associated with clopidogrel resistance (O.R. 5.42 [95% CI 1.82-16.11]). This risk factor was independent of the gender effect. CONCLUSIONS: This is the first large study in clopidogrel treated patients suggesting that a homozygote H2 genotype contributes to clopidogrel resistance. The clinical significance of this finding remains to be demonstrated.

Tirofiban optimizes platelet inhibition for immediate percutaneous coronary intervention in high-risk acute coronary syndromes.

It was the aim of this study to compare the efficacy of early platelet inhibition by 600 mg clopidogrel and acetylsalicylic acid (ASA) to a triple therapy including a glycoprotein IIb-IIIa receptor blocker. Immediate percutaneous coronary intervention (PCI) is recommended for high-risk acute coronary syndromes. In this setting the efficacy of platelet inhibition is unknown. One hundred patients were randomized to receive ASA and 600 mg clopidogrel, or additional open-label tirofiban (bolus of 10 microg/kg body weight followed by continued infusion of 0.15 microg/kg body weight per minute) as soon as non-ST-segment elevation myocardial infarction was diagnosed. The primary endpoint was the reduction of infarct size determined by post-interventional increases of cardiac troponin T (cTnT). Secondary endpoints included platelet function measured by optical and impedance aggregometry using ADP (5 and 20 microM) and collagen (1 microg/ml) as platelet agonists. Tirofiban maximized platelet inhibition (p < 0.0001) immediately and was associated with significantly lower post-interventional cTnT concentrations (p = 0.0352). In the dual platelet inhibition arm, clopidogrel was not effective in 69% of patients at the time of coronary intervention, and still in 47%, if pre-treatment time was >120 minutes. The frequency of cardiovascular (death, myocardial infarction, revascularization) and bleeding events was comparable. Platelet aggregation is not adequately inhibited in cTnT-positive patients in the setting of immediate PCI with very short pre-treatment times. Only tirofiban provided consistent and effective inhibition of platelet aggregation at the time of immediate or very early invasive therapy.

Highly sensitive cardiac troponin T values remain constant after brief exercise- or pharmacologic-induced reversible myocardial ischemia.

BACKGROUND: Using a new precommercial high-sensitivity cardiac troponin T (hsTnT) assay, we evaluated whether hsTnT increases after reversible myocardial ischemia. METHODS: In 195 patients undergoing nuclear stress testing (ST) using single-photon emission computed tomography (SPECT) for suspected ischemic heart disease, we measured hsTnT before and 18 min, 4 h, and 24 h after the stress test. Thirty patients were excluded before ST because of cardiac troponin T (cTnT) >30 ng/L (0.03 mug/L) as measured by the fourth-generation commercial test. Another 65 patients were excluded because of a combination of fixed and reversible perfusion defects (PDs) after SPECT. RESULTS: We studied 18 patients with reversible PDs, 41 patients with fixed PDs, and 41 patients without any PDs. Of these 100 patients, 61 received dynamic ST and 39 pharmacological ST. Median baseline hsTnT concentrations (25th, 75th percentile) were comparable in patients with reversible, fixed, and no PDs [5.57 (2.47, 12.60), 8.01 (4.55, 12.44), and 6.90 (4.63, 10.59) ng/L, respectively]. After ST, median hsTnT concentrations did not change in the reversible, fixed, or no PD groups from baseline to 18 min [-0.41 (-0.81, 0.01), 0.01 (-0.75, 0.79), and 0.36 (-0.42, 1.01) ng/L] or from baseline to 4 h [-0.56 (-1.82, 0.74), 0.24 (-0.60, 1.45), and 0.23 (-0.99, 1.15) ng/L]. Median baseline hsTnT concentrations tended to be higher in patients undergoing pharmacological vs dynamic ST; however, there were no significant increases in hsTnT concentrations after either type of ST. CONCLUSIONS: Elevation of cTnT is rather a consequence of irreversible myocyte death than reversible myocardial ischemia after exercise or pharmacologic myocardial ischemia.

Cardiac magnetic resonance imaging study for quantification of infarct size comparing directly serial versus single time-point measurements of cardiac troponin T.

OBJECTIVES: We compared single-point cardiac troponin T (cTnT) measurements with parameters from serial sampling during 96 h after acute myocardial infarction with magnetic resonance imaging measured infarct mass. BACKGROUND: Contrast-enhanced magnetic resonance imaging (CE-MRI) allows exact quantification of myocardial infarct size. Clinically, measurement of cardiac biomarkers is a more convenient alternative. METHODS: The CE-MRI infarct mass was determined 4 days after primary percutaneous coronary intervention in 31 ST-segment elevation myocardial infarction (STEMI) and 30 non-ST-segment elevation myocardial infarction (NSTEMI) patients. All single-point, peak, and integrated area under the curve (AUC) cTnT values were plotted against CE-MRI infarct mass. RESULTS: All single-point and serial cTnT values were significantly higher in STEMI than in NSTEMI (p < 0.01) patients. Except for the admission values, all single-point values on any of the first 4 days, peak cTnT and AUC cTnT were found to correlate comparably well with infarct mass. Among single-point measurements, cTnT on day 4 (cTnTD4) showed highest correlation and performed as well as peak cTnT or AUC cTnT (r = 0.66 vs. r = 0.65 vs. r = 0.69). Receiver-operator characteristic analysis demonstrated that cTnTD4 >0.84 microg/l predicted infarct mass above median as well as peak cTnT >1.57 microg/l or AUC cTnT (receiver-operator characteristic for AUC: 0.839 vs. 0.866 vs. 0.893). However, estimation of infarct mass with cTnTD4, peak cTnT, and AUC cTnT was worse in patients with NSTEMI (r = 0.36, r = 0.5, r = 0.36) than in STEMI (r = 0.75 vs. r = 0.65 vs. r = 0.76). CONCLUSIONS: All single-point cTnTs, except on admission, give a good estimation of infarct size and perform as well as peak cTnT or AUC cTnT. Infarct estimation by single-point measurements, particularly cTnTD4, may gain clinical acceptance because the measurement is easy and inexpensive.

Platelet-associated LIGHT (TNFSF14) mediates adhesion of platelets to human vascular endothelium.

LIGHT (TNFSF 14) belongs to the tumor necrosis factor super-family and is expressed by different types of immune cells. Recently, LIGHT was found to be associated with platelets and released upon activation. Activation of endothelial cells by recombinant LIGHT results in pro-inflammatory and pro-thrombotic changes, qualitatively comparable to effects of CD40 ligand. Given the important role of platelet-associated CD40 ligand in vascular inflammatory responses we investigated the role of LIGHT for activation of endothelium and adhesion of platelets to endothelial cells. Expression of LIGHT was detected on thrombocytes upon exposure to ADP or TRAP-1. The expression of the LIGHT receptors TR2 and LTbetaR on native human endothelial cells was confirmed by FACS analysis. LIGHT mediated adhesion of platelets to endothelium significantly, occurring both under static and dynamic flow conditions. This interaction was inhibited by a monoclonal antibody to LIGHT but not a control IgG. Moreover, in-vitro stimulation of endothelial cells with recombinant soluble human LIGHT (rhLIGHT) resulted in significantly increased transcriptional and translational upregulation of inflammatory markers ICAM-1, tissue factor (TF) and IL-8. This activation of endothelial cells by LIGHT was mediated by NFkappaB activation and qualitatively comparable to that induced by membrane-bound CD40-ligand on transfected cells. Furthermore, plasma levels of patients with myocardial infarction, in those with ST-elevation myocardial infarction (STEMI), showed increased plasma levels of LIGHT compared with healthy controls. In conclusion, platelet-associated LIGHT is involved in adhesion of platelets to endothelium while soluble LIGHT induces a pro-inflammatory state in vascular endothelial cells. LIGHT may thus be implicated in the pathogenesis of atherosclerosis and acute coronary syndrome, as evidenced by serum levels.

Quantitative evaluation of myocardial blush to assess tissue level reperfusion in patients with acute ST-elevation myocardial infarction: incremental prognostic value compared with visual assessment.

BACKGROUND: Tissue level reperfusion gauges functional recovery in acute ischemic syndromes. However, its current clinical assessment is based upon visual interpretation of myocardial blush grade (MBG), which is operator dependent. The purpose of the study was to test whether quantification of MBG can enhance the predictive value of visual assessment for functional recovery in patients with acute ST-elevation myocardial infarction (STEMI). METHODS: Myocardial blush grade was assessed in 124 consecutive patients with STEMI visually and quantitatively, analyzing the time course of blush intensity rise. We defined Gmax as the peak gray level intensity and Tmax as the time to peak intensity. Ejection fraction >50% at 4 to 6 months of follow-up was deemed as the primary end point for assessment of successful tissue reperfusion. RESULTS: Ejection fraction >50% at follow-up was predicted by visual MBG with moderate sensitivity (65%) and specificity (64%). However, a cutoff value of Gmax/Tmax = 3.1/s yielded significantly higher sensitivity and specificity (91% and 96%, respectively, for both P < .01). Gmax/Tmax was the most powerful predictor of follow-up ejection fraction >50% (relative risk of 4.6 vs 3.2 for visual MBG). CONCLUSIONS: Quantitative MBG is highly predictive for functional recovery in patients with STEMI and provides incremental prognostic value to visual assessment. Thus, this simple approach may be used to gauge reperfusion strategies in acute ischemic syndromes.

Determination of clopidogrel resistance by whole blood platelet aggregometry and inhibitors of the P2Y12 receptor.

BACKGROUND: Inhibition of platelet aggregation by clopidogrel may be insufficient in up to 30% of users. These nonresponders carry an increased risk of cardiovascular events. We reported here a simple assay to study clopidogrel responsiveness. METHODS: Electrical impedance aggregometry was performed in diluted whole blood in the presence of 5 and 20 micromol/L ADP. Some samples were incubated with 0.1 mmol/L methyl-S-adenosine monophosphate (MeSAMP), a P2Y12 receptor blocker, to maximize inhibition of aggregation before aggregometry. To validate the assay, we analyzed 6-min impedance in 21 healthy probands and 244 patients with coronary artery disease (CAD). RESULTS: At 5 micromol/L ADP, the imprecision of the assay was 11%. Mean (SD) impedance of the healthy cohort was 12.2 (2.2) Omega. The mean - 3 SD was used to define the cutoff for clopidogrel responsiveness: responders and nonresponders exhibited a 6-min impedance < or =5 Omega and >5 Omega, respectively. Samples from nonresponders were incubated with MeSAMP and analyzed again to distinguish pharmacokinetic and pharmacodynamic types of resistance. Sixteen percent of CAD patients were classified as nonresponders (38 and 2 cases of pharmacokinetic and pharmacodynamic resistance, respectively). Female sex was strongly associated with clopidogrel resistance (P = 0.0002, Fisher exact test). A higher clopidogrel loading dose (P = 0.0353, Mann-Whitney U-test) was given to responders (median, 450 mg) than nonresponders (median, 300 mg). Age and cardiovascular diagnosis showed no significant associations. CONCLUSIONS: Impedance aggregometry using 5 mumol/L ADP is a useful tool for studying clopidogrel responsiveness. MeSAMP allows characterization of responsiveness "on treatment" and may be useful for optimizing clopidogrel dosing.

[Diagnosis of ischemia]. / Ischämiediagnostik.

Clinically, coronary artery disease (CAD) presents either as stable angina pectoris or as an acute coronary syndrome. Atypical chest pain or silent myocardial ischemia is not uncommon and may obscure the diagnosis of CAD. Whereas primary or secondary prevention is indicated in all cases with suspected or documented atherosclerosis, cardiac catheterization and revascularization therapy is restricted to patients with significant CAD. In order to detect hemodynamically significant coronary artery stenoses, exercise stress tests or noninvasive stress imaging are usually implemented in the diagnostic workup. By contrast, patients presenting with an acute coronary syndrome require an initial risk stratification for estimation of the acute thrombotic risk associated with the underlying vulnerable plaque. Consecutively, patients without high-risk features need a thorough noninvasive evaluation for the presence of significant CAD which is comparable to patients presenting with chronic stable angina pectoris.