Impact of On-Clopidogrel Platelet Reactivity on Incidence of Peri-Interventional Bleeding in Patients Undergoing Transcatheter Aortic Valve Implantation.

Dual anti-platelet therapy (DAPT) with clopidogrel and acetylsalicylic acid (ASA) has previously been recommended after transcatheter aortic valve implantation (TAVI) and is still the standard of care in patients who underwent coronary stent placement within 3 months prior to TAVI. This study sought to evaluate whether on-treatment platelet reactivity is a predictor for the occurrence of bleeding events after TAVI. This study enrolled 484 patients undergoing TAVI from November 2013 until April 2018. Patients were either on long-term DAPT with clopidogrel and ASA or received loading doses of both drugs before TAVI, reflecting the standard of care at the time of the patient's enrollment. Platelet reactivity was determined by multi-electrode impedance aggregometry before TAVI, at days 1 and 5 thereafter. Peri-interventional bleeding was assessed up to 5 days following TAVI and coded according to BARC-classification. Bleeding events were seen in 199 (41.1%) patients. The most frequent were BARC 2 bleeding cases (24.2%), followed by BARC 1 (6.0%), BARC 3b (5.2%), and BARC 3a (4.5%) cases. Low on-clopidogrel platelet reactivity before TAVI was present in 243 patients, of which 44.4% had a bleeding event. In contrast, the incidence of bleeding was 30.5% in the 95 patients with high on-clopidogrel platelet reactivity. Multivariate logistic regression analysis identified low/normal/high on-clopidogrel platelet reactivity (OR: 0.533; CI: 0.309-0.917; p = 0.023) and use of oral anticoagulation (OR: 1.766; CI: 1.209-2.581; p = 0.003) as strongest predictors for peri-interventional bleeding events. These findings support current recommendations advocating against the routine use of dual antiplatelet therapy following TAVI.

Impact of high platelet turnover on the platelet transcriptome: Results from platelet RNA-sequencing in patients with sepsis.

BACKGROUND: Sepsis is associated with high platelet turnover and elevated levels of immature platelets. Changes in the platelet transcriptome and the specific impact of immature platelets on the platelet transcriptome remain unclear. Thus, this study sought to address whether and how elevated levels of immature platelets affect the platelet transcriptome in patients with sepsis. METHODS: Blood samples were obtained from patients with sepsis requiring vasopressor therapy (n = 8) and from a control group of patients with stable coronary artery disease and otherwise similar demographic characteristics (n = 8). Immature platelet fraction (IPF) was determined on a Sysmex XE 2100 analyser and platelet function was tested by impedance aggregometry. RNA from leukocyte-depleted platelets was used for transcriptome analysis by Next Generation Sequencing integrating the use of unique molecular identifiers. RESULTS: IPF (median [interquartile range]) was significantly elevated in sepsis patients (6.4 [5.3-8.7] % vs. 3.6 [2.6-4.6] %, p = 0.005). Platelet function testing revealed no differences in adenosine diphosphate- or thrombin receptor activating peptide-induced platelet aggregation between control and sepsis patients. Putative circular RNA transcripts were decreased in platelets from septic patients. Leukocyte contamination defined by CD45 abundance levels in RNA-sequencing was absent in both groups. Principal component analysis of transcripts showed only partial overlap of clustering with IPF levels. RNA sequencing showed up-regulation of 524 and down-regulation of 118 genes in platelets from sepsis patients compared to controls. Upregulated genes were mostly related to catabolic processes and protein translation. Comparison to published platelet transcriptomes showed a large overlap of changes observed in sepsis and COVID-19 but not with reticulated platelets from healthy donors. CONCLUSIONS: Patients with sepsis appear to have a less degraded platelet transcriptome as indicated by increased levels of immature platelets and decreased levels of putative circular RNA transcripts. The present data suggests that increased protein translation is a characteristic mechanism of systemic inflammation.

Prevalence and Causes of Myocardial Infarction with Non-Obstructive Coronary Arteries in a Contemporary Cohort of Patients with Suspected Myocardial Infarction.

BACKGROUND: A significant proportion of patients presenting with acute myocardial infarction (MI) has no coronary obstruction at coronary angiography and no other obvious non-coronary pathophysiology causing MI. These patients are classified as MI with non-obstructive coronary arteries (MINOCA). Data on incidence and predictors of MINOCA are still limited. METHODS: This study enrolled patients presenting symptoms suggestive of MI and undergoing a comprehensive cardiac work-up including an early invasive strategy. Patients with non-obstructive coronary arteries and without other obvious reasons for MI were scheduled for further work-up including magnetic resonance or intraluminal imaging. MINOCA was diagnosed according to the current European Society of Cardiology guidelines. RESULTS: From the 1532 patients enrolled, 730 had available coronary imaging and 546 were diagnosed with MI. No significant coronary obstructions were found in 117 patients with MI. After the exclusion of 6 patients with acute myocarditis or takotsubo-syndrome as well as 88 with type II MI, 23 patients were diagnosed with MINOCA (4% of all MIs). Among these 23 patients, the most common etiology of MINOCA was thromboembolic events followed by coronary spasm. Female sex, the absence of hypercholesterolemia, and a normal left-ventricular ejection fraction were independently predictive for MINOCA compared to patients with other causes of MI. CONCLUSION: More than 20% of patients presenting with acute MI showed no significant coronary obstruction. About 4% of these patients were diagnosed with MINOCA. Female sex, a lower cardiovascular risk profile, and normal left-ventricular function were predictive for MINOCA.

Ultrastructural, transcriptional, and functional differences between human reticulated and non-reticulated platelets.

BACKGROUND: Reticulated platelets (RP) are the youngest circulating platelets in blood. An increased amount of this subpopulation is associated with higher cardiovascular risk and mortality. OBJECTIVES: It is unknown to what extent intrinsic properties of RP contribute to their hyperreactive features. This study is the first providing a multifactorial approach based on ultrastructural, transcriptional, and functional analysis of RP compared to non-RP sorted by flow cytometry. METHODS: Reticulated platelets and non-RP were sorted after platelet staining with SYTO 13. Employing transmission electron microscopy, 1089 micrographs were analyzed for platelet size, amounts of intracellular structures, and anatomical surrogates indicating activation. Long and small RNA-sequencing (RNA-seq) were performed for analyzing differential gene expression. Functional analysis of P-selectin-an upregulated mRNA in RP-was performed in healthy subjects and patients on P2Y12 -inhibitors. RESULTS: Electron micrographs uncovered distinct ultrastructural differences in RP versus non-RP. Cross sections were 1.9-fold larger in RP (P < .0001). Amounts of α-granules, dense granules, open canalicular system-openings, and mitochondria were increased in RP, which persisted after adjustment for platelet size. Long RNA-seq showed 1212 upregulated transcripts that are predominantly associated to platelet shape change, aggregation, and activation; 1264 mRNAs were downregulated in RP. Small RNA-seq did not reveal any differentially expressed transcripts. Functional analysis displayed higher P-selectin expression as compared to non-RP upon ADP- or TRAP-stimulation. CONCLUSIONS: Our results demonstrate that altered intrinsic structural and molecular properties contribute to the hyperreactivity of RP. These properties and an increased amount of RP may account for the association with cardiovascular risk.

Need for pacemaker implantation in patients with normal QRS duration immediately after transcatheter aortic valve implantation.

AIMS: We sought to assess the need for permanent pacemaker implantation (PPI) in patients with QRS <120 ms in electrocardiogram (ECG) after transcatheter aortic valve implantation (TAVI). METHODS AND RESULTS: We retrospectively analysed 1139 consecutive patients who underwent transfemoral TAVI between 2008 and 2016, receiving different valve types. All patients were surveyed by continuous ECG monitoring for 48 h, 12-lead ECGs starting immediately after procedure, as well as 24-h Holter recording the day before discharge. Indication for PPI was at the discretion of the attending physician. Among 760 patients with QRS <120 ms prior to the TAVI procedure, 400 patients showed QRS <120 ms immediately after procedure, whereas 360 patients had QRS ≥120 ms. In the group with QRS <120 ms, PPI was performed in 34 patients [8.5%; 95% confidence interval (CI) 5.6-11.2%] during the first week. Eight of the PPIs in the group with QRS <120 ms (2%; CI 0.8-3.5%) fulfilled Class I indications for PPI after TAVI, whereas 26 PPIs had different indications [left bundle branch block, sick sinus, low-grade atrioventricular (AV) block]. Complete AV block developed in three patients of the group of QRS <120 ms (0.75%; CI 0.0-1.7%), which in all cases occurred after the 48 h-surveillance period. During 1-year follow-up, 11 PPIs were performed (2.8%; CI 1.2-4.5%), thereof three PPI for Class I indications including one complete AV block. CONCLUSION: In patients with QRS duration <120 ms immediately after TAVI, the risk for complete AV block was low during the first week after TAVI and 1-year follow-up.

Antagonizing P2Y12 Receptor Inhibitors: Current and Future Options.

There are clinical scenarios where the balance between the risk of ischemic and bleeding events leads to the clinical decision to reverse the antiplatelet effect of P2Y12 receptor inhibitors. These scenarios comprise emergency situations such as active severe bleeding, urgent procedures with presumed high bleeding risk, or major trauma with (anticipated) bleeding. Supplementation of platelets has been investigated in ex vivo as well as in in vivo studies. These studies indicate that the inhibition of adenosine diphosphate-induced aggregation by the irreversibly binding thienopyridine derivatives clopidogrel and prasugrel can be reversed by administration of platelet concentrates. Supplementation of platelets in patients on prasugrel is more effective if this can be transfused > 6 hours after last dosing. Studies on the reversal effect obtained by administration of platelet concentrates in patients on ticagrelor show conflicting results. Experimental data suggest that administration of serum albumin might increase the reversal effect. A monoclonal antibody fragment (PB2452) for neutralizing ticagrelor is currently in clinical development. A recently published first in man study shows that reversal of platelet inhibition occurs within 5 minutes after start of administration and the effect is maintained for 20 to 24 hours after a 16-hour infusion which is by far the most effective approach for reversal of ticagrelor.

Evaluation of an Alternative Staining Method Using SYTO 13 to Determine Reticulated Platelets.

Reticulated platelets reflect the rate of platelet turnover and represent the youngest circulating platelets in peripheral blood. Reticulated platelets contain residual ribonucleic acid (RNA) from megakaryocytes which is lost in a time-dependent manner and can be transcribed into proteins even in the absence of a nucleus. An increased proportion of reticulated platelets is associated with higher platelet reactivity, cardiovascular events and mortality. At present, a fully automated assay system (SYSMEX haematology analyser) is available for analysis. This method, however, is not suitable for extended laboratory investigations like subsequent cell sorting. Flow cytometry analysis after staining with thiazole orange (TO) is frequently used in such settings despite several limitations. Here, we describe a new assay for determination of reticulated platelets by flow cytometry using the nucleic acid staining dye SYTO 13 and compare it with SYSMEX and TO staining as current standards. A significant correlation between immature platelet fraction (IPF) determined by SYSMEX XE-2100 analyser and results obtained with the SYTO 13-based assay was observed (r = 0.668, p < 0.001) which was stable during a reasonable time period. In contrast, the correlation between TO staining and IPF was weaker (r = 0.478, p = 0.029) and lost after 90 minutes of staining. SYTO 13 staining of platelets enabled sorting of RNAlow and RNArich platelets which was confirmed by RNA quantification of sorted platelets. Except for fixation of platelets, sorting of these platelet sub-populations was stable under various experimental settings. In summary, determination of reticulated platelets with the new SYTO 13 assay offers distinct technical advantages enabling further laboratory processing.

Effect of mineralocorticoid receptor antagonists on cardiac function in patients with heart failure and preserved ejection fraction: a systematic review and meta-analysis of randomized controlled trials.

Heart failure with preserved ejection fraction (HFpEF) is a disease with limited evidence-based treatment options. Mineralocorticoid receptor antagonists (MRA) offer benefit in heart failure with reduced ejection fraction (HFrEF), but their impact in HFpEF remains unclear. We therefore evaluated the effect of MRA on echocardiographic, functional, and systemic parameters in patients with HFpEF by a systematic review and meta-analysis. We searched MEDLINE, EMBASE, clinicaltrials.gov , and Cochrane Clinical Trial Collection to identify randomized controlled trials that (a) compared MRA versus placebo/control in patients with HFpEF and (b) reported echocardiographic, functional, and/or systemic parameters relevant to HFpEF. Studies were excluded if: they enrolled asymptomatic patients; patients with HFrEF; patients after an acute coronary event; compared MRA to another active comparator; or reported a follow-up of less than 6 months. Primary outcomes were changes in echocardiographic parameters. Secondary end-points were changes in functional capacity, quality of life measures, and systemic parameters. Quantitative analysis was performed by generating forest plots and calculating effect sizes by random-effect models. Between-study heterogeneity was assessed through Q and I2 statistics. Nine trials with 1164 patients were included. MRA significantly decreased E/e' (mean difference - 1.37, 95% confidence interval - 1.72 to - 1.02), E/A (- 0.04, - 0.08 to 0.00), left ventricular end-diastolic diameter (- 0.78 mm, - 1.34 to - 0.22), left atrial volume index (- 1.12 ml/m2, - 1.91 to - 0.33), 6-min walk test distance (- 11.56 m, - 21 to - 2.13), systolic (- 4.75 mmHg, - 8.94 to - 0.56) and diastolic blood pressure (- 2.91 mmHg, - 4.15 to - 1.67), and increased levels of serum potassium (0.23 mmol/L, 0.19 to 0.28) when compared with placebo/control. In patients with HFpEF, MRA treatment significantly improves indices of cardiac structure and function, suggesting a decrease in left ventricular filling pressure and reverse cardiac remodeling. MRA increase serum potassium and decrease blood pressure; however, a small decrease in 6-min-walk distance is also noted. Larger prospective studies are warranted to provide definitive answers on the effect of MRA in patients with HFpEF.

Impact of On-Clopidogrel Platelet Reactivity on Incidence of Hypoattenuated Leaflet Thickening After Transcatheter Aortic Valve Replacement.

OBJECTIVES: To assess the impact of on-clopidogrel platelet reactivity (PR) on HALT, the authors prospectively tested whether patients with below-median on-clopidogrel PR have a lower incidence of HALT compared with those with above-median on-clopidogrel PR. BACKGROUND: It is unclear whether the apparent ineffectiveness of clopidogrel in preventing hypoattenuated leaflet thickening (HALT) after transcatheter aortic valve replacement (TAVR) questions the concept of P2Y12 inhibition after TAVR or is a consequence of an inadequate response to clopidogrel in elderly patients with severe aortic stenosis. METHODS: Patients were either on long-term dual antiplatelet therapy with clopidogrel and acetylsalicylic acid or were given bolus doses of both drugs the day before TAVR. Adenosine diphosphate (ADP)-induced multielectrode impedance aggregometry was performed before TAVR. After TAVR, clopidogrel was continued in all patients. Computed tomographic angiography was performed to detect HALT. RESULTS: Of 331 patients enrolled, computed tomographic angiography was performed in 200 at 5 days (interquartile range: 4 to 6 days). Among patients with below-median ADP-induced PR (<180 AU · min), 16 were diagnosed with HALT, whereas 20 patients with above-median PR were diagnosed with HALT (p = 0.58). Among patients with high on-clopidogrel PR (>468 AU · min; n = 29), 7 (24%) displayed HALT, compared with 19 (17%) with ADP-induced PR ≤468 AU · min (p = 0.43). Consistently, ADP-induced PR as a continuous variable was not significantly associated with HALT (p = 0.75). Oral anticoagulation was associated with reduced rates of HALT (odds ratio: 0.41; 95% CI: 0.18 to 0.96; p = 0.04). CONCLUSIONS: On-clopidogrel ADP-induced PR was not significantly associated with the occurrence of HALT. In contrast, oral anticoagulation was associated with reduced rates of HALT.

Long-term results after PCI of unprotected distal left main coronary artery stenosis: the Bifurcations Bad Krozingen (BBK)-Left Main Registry.

AIMS: Percutaneous coronary intervention (PCI) of unprotected distal left main stenosis (UDLM) is increasingly performed as an alternative to surgical treatment. The optimal strategy for stenting in this setting is still a matter of debate. Therefore, this analysis investigated the long-term clinical outcome of a single- versus a double-stenting strategy for treatment of UDLM. METHODS AND RESULTS: From a large registry, 867 consecutive patients with UDLM undergoing either single or double stenting with drug-eluting stents (DES) were identified. Follow-up was up to 10 (median 3.1, interquartile range 1.1-5.3) years. Primary endpoint was MACE consisting of all-cause death, myocardial infarction, or target lesion re-intervention (TLR). Secondary clinical endpoints included these single endpoints and stent thrombosis. MACE occurred in 41.5% after single and in 49.0% after double stenting (P = 0.03). TLR was lower after single (17.4%) as compared to double stenting (27.2%; P < 0.01). Between single and double stenting, there were no significant differences for death (26.4 versus 23.3%; P = 0.31), death or myocardial infarction (29.1 versus 27.2%; P = 0.55), or definite/probable stent thrombosis (1.3 versus 2.1%; P = 0.42). CONCLUSIONS: Compared with single stenting, double stenting was associated with a significantly higher long-term risk of MACE. This was driven by a higher incidence of TLR, whereas the risk of death, MI, or stent thrombosis was similar between the two strategies.

Uncontrolled Diabetes Mellitus Has No Major Influence on the Platelet Transcriptome.

BACKGROUND: Diabetes mellitus (DM) has been associated with increased platelet reactivity as well as increased levels of platelet RNAs in plasma. Here, we sought to evaluate whether the platelet transcriptome is altered in the presence of uncontrolled DM. METHODS: Next-generation sequencing (NGS) was performed on platelet RNA for 5 patients with uncontrolled DM (HbA1c 9.0%) and 5 control patients (HbA1c 5.5%) with otherwise similar clinical characteristics. RNA was isolated from leucocyte-depleted platelet-rich plasma. Libraries of platelet RNAs were created separately for long RNAs after ribosomal depletion and for small RNAs from total RNA, followed by next-generation sequencing. RESULTS: Platelets in both groups demonstrated RNA expression profiles characterized by absence of leukocyte-specific transcripts, high expression of well-known platelet transcripts, and in total 6,343 consistently detectable transcripts. Extensive statistical bioinformatic analysis yielded 12 genes with consistently differential expression at a lenient FDR < 0.1, thereof 8 protein-coding genes and 2 genes with known expression in platelets (MACF1 and ITGB3BP). Three of the four differentially expressed noncoding genes were YRNAs (RNY1, RNY3, and RNY4) which were all downregulated in DM. 23 miRNAs were differentially expressed between the two groups. Of the 13 miRNAs with decreased expression in the diabetic group, 8 belonged to the DLK1-DIO3 gene region on chromosome 14q32.2. CONCLUSIONS: In this study, uncontrolled DM had a remote impact on different components of the platelet transcriptome. Increased expression of MACF1, together with supporting predicted mRNA-miRNA interactions as well as reduced expression of RNYs in platelets, may reflect subclinical platelet activation in uncontrolled DM.

A Randomized Trial to Assess Regional Left Ventricular Function After Stent Implantation in Chronic Total Occlusion: The REVASC Trial.

OBJECTIVES: The aim of this study was to investigate whether percutaneous coronary intervention (PCI) of chronic total occlusions (CTOs) improves left ventricular function. BACKGROUND: The benefit of PCI in CTOs is still controversial. METHODS: Patients with CTOs who were candidates for PCI were eligible for the study and were randomized to PCI or no PCI of CTO. Relevant coexisting non-CTO lesions were treated as indicated. Patients underwent cardiac magnetic resonance imaging at baseline and at 6 months. The primary endpoint was the change in segmental wall thickening (SWT) in the CTO territory. Secondary endpoints were improvement of regional wall motion and changes in left ventricular volumes and ejection fraction. Furthermore, major adverse coronary events after 12 months were assessed. RESULTS: The CTO PCI group comprised 101 patients and the no CTO PCI group 104 patients. The change in SWT did not differ between the CTO PCI (4.1% [interquartile range: 14.6 to 19.3]) and no CTO PCI (6.0% [interquartile range: 8.6 to 6.0]) groups (p = 0.57). Similar results were obtained for other indexes of regional and global left ventricular function. Subgroup analysis revealed that only in patients without major non-CTO lesions (basal SYNTAX [Synergy Between Percutaneous Coronary Intervention With Taxus and Cardiac Surgery] score ≤13) CTO PCI was associated with larger improvement in SWT than no CTO PCI (p for interaction = 0.002). Driven by repeat intervention, major adverse coronary event rates at 12 months were significantly lower in the CTO PCI group (16.3% vs. 5.9%; p = 0.02). CONCLUSIONS: No benefit was seen for CTO PCI in terms of the primary endpoint, SWT, or other indexes of left ventricular function. CTO PCI resulted in clinical benefit over no CTO PCI, as evidenced by reduced major adverse coronary event rates at 12 months.

Efficacy and safety of bivalirudin for percutaneous coronary intervention in acute coronary syndromes: a meta-analysis of randomized-controlled trials.

AIMS: The efficacy and safety of bivalirudin in patients undergoing percutaneous coronary intervention (PCI) for treatment of acute coronary syndromes (ACS) remains controversial despite recent evidence from large randomized-controlled trials (RCTs). Thus, this systematic review and meta-analysis sought to investigate the efficacy and safety of bivalirudin as compared to heparin in patients with ACS undergoing PCI. METHODS AND RESULTS: Medline/PubMed, Cochrane Central Register of Controlled Trials, and Clinical Trials.gov databases were searched for RCTs. Primary endpoint was MACE consisting of all-cause death, myocardial infarction, and stroke within 30 days. Secondary endpoints were components of the primary endpoint and stent thrombosis. The primary safety endpoint was major bleeding. We identified 12 RCTs comprising 33,844 patients. Between bivalirudin and heparin, there were no significant differences for MACE (OR 1.06; 95% CI 0.96-1.17; p = 0.24), death, myocardial infarction, and stent thrombosis. Similar results were seen following stratification by use of glycoprotein inhibitors (GPI). Major bleeding trended to be less frequent in patients treated with bivalirudin. However, no safety benefit for bivalirudin was seen when use of GPI was balanced between groups (OR 0.88; 95% CI 0.67-1.16; p = 0.35; p for heterogeneity < 0.01). CONCLUSIONS: Compared with heparin, bivalirudin was associated with a similar incidence of ischemic events following PCI for ACS. An association of bivalirudin with decreased bleeding was not seen with balanced use of GPI.

Distinct epigenetic programs regulate cardiac myocyte development and disease in the human heart in vivo.

Epigenetic mechanisms and transcription factor networks essential for differentiation of cardiac myocytes have been uncovered. However, reshaping of the epigenome of these terminally differentiated cells during fetal development, postnatal maturation, and in disease remains unknown. Here, we investigate the dynamics of the cardiac myocyte epigenome during development and in chronic heart failure. We find that prenatal development and postnatal maturation are characterized by a cooperation of active CpG methylation and histone marks at cis-regulatory and genic regions to shape the cardiac myocyte transcriptome. In contrast, pathological gene expression in terminal heart failure is accompanied by changes in active histone marks without major alterations in CpG methylation and repressive chromatin marks. Notably, cis-regulatory regions in cardiac myocytes are significantly enriched for cardiovascular disease-associated variants. This study uncovers distinct layers of epigenetic regulation not only during prenatal development and postnatal maturation but also in diseased human cardiac myocytes.

Alteration of von Willebrand Factor after Transcatheter Aortic Valve Replacement in the Absence of Paravalvular Regurgitation.

Moderate or severe paravalvular regurgitation after transcatheter aortic valve implantation (TAVI) is frequently associated with a loss of high-molecular-weight multimers of von Willebrand factor (VWF) and a reduced VWF collagen-binding capacity. It is unclear whether this phenomenon can also be observed in patients with mild paravalvular regurgitation, and whether there are differences between patients undergoing conventional aortic valve replacement (AVR) or TAVI. We analysed the multimeric structure of VWF and the ratio of VWF collagen-binding capacity to VWF antigen pre- and postoperatively in 12 patients scheduled for AVR and in 31 patients scheduled for TAVI. Echocardiographic examinations were performed pre-, intra- and postoperatively. Nine patients (75%) undergoing AVR and 18 patients (58%) undergoing TAVI showed pathological VWF functionality preoperatively (p = 0.48). Five to 7 days postoperatively, VWF functionality normalised in all patients with AVR, four of them with mild paravalvular regurgitation. VWF functionality was still altered in nine patients after TAVI (p = 0.044 between groups), five of them with and four without mild paravalvular regurgitation (p = 0.1).Altered VWF functionality was observed in nearly one-third of patients after TAVI, but not after AVR. This phenomenon was not related to paravalvular regurgitation, but may indicate differences in the response of the haemostatic system to the prosthetic heart valve design or the valve replacement procedure.

DNA methylation signatures follow preformed chromatin compartments in cardiac myocytes.

Storage of chromatin in restricted nuclear space requires dense packing while ensuring DNA accessibility. Thus, different layers of chromatin organization and epigenetic control mechanisms exist. Genome-wide chromatin interaction maps revealed large interaction domains (TADs) and higher order A and B compartments, reflecting active and inactive chromatin, respectively. The mutual dependencies between chromatin organization and patterns of epigenetic marks, including DNA methylation, remain poorly understood. Here, we demonstrate that establishment of A/B compartments precedes and defines DNA methylation signatures during differentiation and maturation of cardiac myocytes. Remarkably, dynamic CpG and non-CpG methylation in cardiac myocytes is confined to A compartments. Furthermore, genetic ablation or reduction of DNA methylation in embryonic stem cells or cardiac myocytes, respectively, does not alter genome-wide chromatin organization. Thus, DNA methylation appears to be established in preformed chromatin compartments and may be dispensable for the formation of higher order chromatin organization.

5'-Hydroxymethylcytosine Precedes Loss of CpG Methylation in Enhancers and Genes Undergoing Activation in Cardiomyocyte Maturation.

BACKGROUND: Cardiomyocytes undergo major changes in DNA methylation during maturation and transition to a non-proliferative state after birth. 5'-hydroxylation of methylated cytosines (5hmC) is not only involved in DNA loss of CpG methylation but is also thought to be an epigenetic mark with unique distribution and functions. Here, we sought to get insight into the dynamics of 5'-hydroxymethylcytosine in newborn and adult cardiomyocytes. METHODS: Cardiomyocyte nuclei from newborn and adult C57BL/6 mice were purified by flow cytometric sorting. 5hmC-containing DNA was captured by selective chemical labeling, followed by deep sequencing. Sequencing reads of library replicates were mapped independently (n = 3 for newborn, n = 2 for adult mice) and merged for further analysis steps. 5hmC coverage was normalized to read length and the total number of mapped reads (RPKM). MethylC-Seq, ChIP-Seq and RNA-Seq data sets of newborn and adult cardiomyocytes served to elucidate specific features of 5hmC at gene bodies and around low methylated regions (LMRs) representing regulatory genomic regions with enhancer function. RESULTS: 163,544 and 315,220 5hmC peaks were identified in P1 and adult cardiomyocytes, respectively. Of these peaks, 66,641 were common between P1 and adult cardiomyocytes with more than 50% reciprocal overlap. P1 and adult 5hmC peaks were overrepresented in genic features such as exons, introns, 3'- and 5'-untranslated regions (UTRs), promotors and transcription end sites (TES). During cardiomyocyte maturation, 5hmC was found to be enriched at sites of subsequent DNA loss of CpG methylation such as gene bodies of upregulated genes (i.e. Atp2a2, Tnni3, Mb, Pdk4). Additionally, centers of postnatally established enhancers were premarked by 5hmC before DNA loss of CpG methylation. CONCLUSIONS: Simultaneous analysis of 5hmC-Seq, MethylC-Seq, RNA-Seq and ChIP-Seq data at two defined time points of cardiomyocyte maturation demonstrates that 5hmC is positively associated with gene expression and decorates sites of subsequent DNA loss of CpG methylation.

Temporal variability in the antiplatelet effects of clopidogrel and aspirin after elective drug-eluting stent implantation. An ADAPT-DES substudy.

Given conflicting data on temporal variability in pharmacodynamic platelet responses to clopidogrel, we investigated platelet reactivity on clopidogrel and aspirin for up to six months after elective percutaneous coronary intervention (PCI) with drug-eluting stents. Platelet reactivity was determined in 102 patients before loading with clopidogrel and aspirin, and on maintenance therapy after PCI on day 1, at one month and six months by VerifyNow™ P2Y12 and Aspirin assays and by residual platelet aggregation (RPA) on light transmission aggregometry using adenosine diphosphate and arachidonic acid. By VerifyNow testing, median (interquartile range) P2Y12 reaction units (PRU) on clopidogrel were 166 (90-234), 195 (124-257), and 198 (141-252) on day 1, one month and six months after PCI, respectively (p=0.005 day 1 to 1 month, and p=0.86 1 month to 6 months). Using a cut-off of > 208 PRU, 35 % of patients had high platelet reactivity (HPR) to clopidogrel on day 1, 43 % at one month, and 46 % at six months after PCI. Between day 1 and six months after PCI, 38.2 % of patients changed clopidogrel responder status at least once. Other cut-offs and RPA yielded similar results. Platelet inhibition by aspirin was consistent over time with only five patients being characterised as having HPR. Considerable variation in individual on-clopidogrel platelet reactivity was present during both the subacute and the late phases of maintenance therapy after elective PCI. Hence, the utility of contemporary platelet function testing to guide antiplatelet therapy may be limited.

Large platelets but not putative endothelial progenitor cells are associated with low strut coverage after drug-eluting stent implantation.

OBJECTIVES: This study assessed whether different subsets of circulating endothelial and putative endothelial progenitor cells (CEC and EPC) correlate with stent strut coverage (SSC) using second generation optical coherence tomography (OCT). BACKGROUND: Due to the lack of imaging modalities with a resolution down to the magnitude of a few cells, the influence of EPC on endothelialisation of drug-eluting stents has not been assessed in patients. METHODS: In 37 patients, SSC of everolimus-eluting stents was assessed by OCT 5-7months after stent implantation. Different subsets of EPC (CD34(+)KDR(+), CD34(+)KDR(+)CD45(dim), CD133(+), CD3(+)CD31(+)), CEC (CD31(+)CD45(-)CD146(+)), and CD31(+)CD45(-)CD146(-) representing large platelets were analysed by flow cytometry, including viability analyses with 7-AAD. Statistical analysis comprised univariate regression analysis and multivariable models integrating OCT and flow cytometry data as well as clinical variables. RESULTS: SSC and frequency of different cell types were highly comparable with previously published data. EPC defined in part by KDR expression were mostly non-viable. On univariate and in multivariable models, no association between EPC counts and strut coverage was detected. For CD31(+)CD45(-)CD146(-) counts, representing large platelets, an inverse relationship with strut coverage was identified by a multivariable regression model adjusting for age, sex, diabetes mellitus, NYHA and CCS class, CRP, serum triglycerides, glucose and creatinine (beta=-9.42, p=0.006). CONCLUSIONS: There was no significant association between EPC or CEC and healing after drug-eluting stent implantation. Yet, CD31(+)CD45(-)CD146(-) cells were associated with low SSC. These data suggest that large platelets may represent a more important mediator of late stent endothelialisation than EPC.