Left atrial appendage volume is an independent predictor of atrial arrhythmia recurrence following cryoballoon pulmonary vein isolation in persistent atrial fibrillation.

Purpose: Pulmonary vein isolation (PVI) is the cornerstone of atrial fibrillation (AF) ablation in persistent AF (persAF), and cryoballoon PVI emerged as an initial ablation strategy. Symptomatic atrial arrhythmia recurrence following successful PVI in persAF is observed more frequently than in paroxysmal AF. Predictors for arrhythmia recurrence following cryoballoon PVI for persAF are not well described, and the role of left atrial appendage (LAA) anatomy is uncertain. Methods: Patients with symptomatic persAF and pre-procedural cardiac computed tomography angiography (CCTA) images undergoing initial second-generation cryoballoon (CBG2) were enrolled. Left atrial (LA), pulmonary vein (PV) and LAA anatomical data were assessed. Clinical outcome and predictors for atrial arrhythmia recurrence were evaluated by univariate and multivariate regression analysis. Results: From May 2012 to September 2016, 488 consecutive persAF patients underwent CBG2-PVI. CCTA with sufficient quality for measurements was available in 196 (60.4%) patients. Mean age was 65.7 ± 9.5 years. Freedom from arrhythmia was 58.2% after a median follow-up of 19 (13; 29) months. No major complications occurred. Independent predictors for arrhythmia recurrence were LAA volume (HR 1.082; 95% CI, 1.032 to 1.134; p = 0.001) and mitral regurgitation ≥ grade 2 (HR, 2.49; 95% CI 1.207 to 5.126; p = 0.013). LA volumes ≥110.35 ml [sensitivity: 0.81, specificity: 0.40, area under the curve (AUC) = 0.62] and LAA volumes ≥9.75 ml (sensitivity: 0.56, specificity 0.70, AUC = 0.64) were associated with recurrence. LAA-morphology, classified as chicken-wing (21.9%), windsock (52.6%), cactus (10.2%) and cauliflower (15.3%), did not predict outcome (log-rank, p = 0.832). Conclusion: LAA volume and mitral regurgitation were independent predictors for arrhythmia recurrence following cryoballoon ablation in persAF. LA volume was less predictive and correlated with LAA volume. LAA morphology did not predict the clinical outcome. To improve outcomes in persAF ablation, further studies should focus on treatment strategies for persAF patients with large LAA and mitral regurgitation.

DNA methylation profiles within the serotonin transporter gene moderate the association of 5-HTTLPR and cortisol stress reactivity.

The serotonin transporter gene-linked polymorphic region (5-HTTLPR) has been implicated in moderating vulnerability to stress-related psychopathology upon exposure to environmental adversity. A recent meta-analysis suggests a potential biological pathway conveying genotype-dependent stress sensitivity by demonstrating a small, but significant association of 5-HTTLPR and cortisol stress reactivity. An arguably more potent approach to detect larger effects when investigating the 5-HTTLPR stress sensitivity hypothesis is to account for both genetic and epigenetic variation in the serotonin transporter gene (SLC6A4). Here, we applied this approach in an experimental setting. Two hundred healthy adults were exposed to a laboratory stressor (Trier Social Stress Test) and cortisol response patterns were assessed as a function of 5-HTTLPR and DNA methylation profiles in SLC6A4. Specifically, we analyzed 83 CpG sites within a 799-bp promoter-associated CpG island of SLC6A4 using a highly sensitive bisulfite pyrosequencing method. Our results suggest that SLC6A4 methylation levels significantly moderate the association of 5-HTTLPR and cortisol stress reactivity. For individuals displaying low levels of SLC6A4 methylation, the S allele relates to increased cortisol stress reactivity in a dose-dependent fashion accounting for 7-9% of the variance in the endocrine stress response. By contrast, no such effect occurred under conditions of high SLC6A4 methylation, indicating that epigenetic changes may compensate for genotype-dependent differences in stress sensitivity. Studying epigenetic markers may advance gene-environment interaction research on 5-HTTLPR as they possibly capture the net effects of environmental influences relevant for stress-related phenotypes under serotonergic control.

Effects of genetic and early environmental risk factors for depression on serotonin transporter expression and methylation profiles.

The serotonin transporter (SERT) gene-linked polymorphic region (5-HTTLPR) has been implicated in moderating the link between life stress and depression. However, respective molecular pathways of gene-environment (GxE) interaction are largely unknown. Sustained alterations in SERT gene expression profiles, possibly mediated by epigenetic modifications, are a frequent correlate of depression and may thus constitute a putative mediator of GxE interaction. Here, we aimed to investigate joint effects of 5-HTTLPR and self-reported environmental adversity throughout the lifespan (prenatal, early and recent stress/trauma) on in vivo SERT mRNA expression in peripheral blood cells. To further evaluate whether environmentally induced changes in SERT expression are mediated by epigenetic modifications, we analyzed 83 CpG sites within a 799-bp promoter-associated CpG island of the SERT gene using the highly sensitive method of bisulfite pyrosequencing. Participants were 133 healthy young adults. Our findings show that both the 5-HTTLPR S allele and maternal prenatal stress/child maltreatment are associated with reduced in vivo SERT mRNA expression in an additive manner. Remarkably, individuals carrying both the genetic and the environmental risk factors exhibited 32.8% (prenatal stress) and 56.3% (child maltreatment) lower SERT mRNA levels compared with those without any risk factor. Our data further indicated that changes in SERT mRNA levels were unlikely to be mediated by DNA methylation profiles within the SERT CpG island. It is thus conceivable that the persistent changes in SERT expression may in turn relate to altered serotonergic functioning and possibly convey differential disease vulnerability associated with 5-HTTLPR and early adversity.

The serotonin transporter gene-linked polymorphic region (5-HTTLPR) and cortisol stress reactivity: a meta-analysis.

Recent meta-analyses have stimulated an active debate on whether the serotonin transporter gene-linked polymorphic region (5-HTTLPR) is associated with an elevated vulnerability to psychiatric diseases upon exposure to environmental adversity. As a potential mechanism explaining genotype-dependent differences in stress sensitivity, altered stress-induced activation of the hypothalamus-pituitary-adrenal (HPA) axis has been investigated in several experimental studies, with most of these studies comprising small samples. We evaluated the association of 5-HTTLPR genotype and cortisol reactivity to acute psychosocial stress by applying a meta-analytical technique based on eleven relevant data sets (total N=1686), which were identified through a systematic literature search up to October 2011. This meta-analysis indicates a small (d=0.27), but significant association between 5-HTTLPR genotype and HPA-axis reactivity to acute psychosocial stress with homozygous carriers of the S allele displaying increased cortisol reactivity compared with individuals with the S/L and L/L genotype. The latter association was not further moderated by participants' age, sex or the type of stressor. Formal testing revealed no evidence for a substantial selection or publication bias. Our meta-analytical results are consistent with a wide variety of experimental studies indicating a significant association between 5-HTTLPR genotype and intermediate phenotypes related to stress sensitivity. Future studies are needed to clarify the consistency of this effect and to further explore whether altered HPA-axis stress reactivity reflects a potential biological mechanism conveying an elevated risk for the development of stress-related disorders in S allele carriers.

[Cardiac computed tomography and ablation of atrial fibrillation]. / Kardiales CT und Ablation von Vorhofflimmern.

Both cardiac computed tomography (CT) and interventional electrophysiology (EP) have evolved considerably in recent years. Technical improvements in CT have significantly reduced the radiation dose in cardiac applications. This imaging technology plays an important role in preprocedural planning and guidance of the procedures in many EP centers worldwide. Furthermore, CT is the imaging modality of choice to diagnose relevant complications in ablation of atrial fibrillation, e.g. pulmonary vein stenosis or atrioesophageal fistula. In anatomically driven ablation procedures, such as balloon-based procedures in atrial fibrillation, detailed analysis of the relevant cardiac structures is absolutely crucial not only to reduce radiation exposure and procedure times but also to improve ablation success and to reduce the occurrence of complications. Current software applications enable 3-dimensional reconstruction of cardiac images and the integration into electroanatomical navigation systems. This article reviews the available evidence in this field and highlights recent developments in image guidance for ablation of atrial fibrillation.

Cryoballoon in atrial fibrillation ablation.

Encouraging results of ablation therapy in patients with paroxysmal atrial fibrillation (AF) have prompted changes in professional practice guidelines. The most recent European guidelines have suggested that ablation might be offered as first-line therapy in selected patients. Cryoballoon ablation is a promising technology in interventional AF therapy. Two different sizes of the cryoballoon are currently available: a smaller (23 mm) and a larger (28 mm) balloon relative to the ostial diameter of the pulmonary veins. New tools, the circular mapping catheter and the use of intracardiac echocardiography, provide important periprocedural information. A meta-analysis of previous studies revealed outcome data with an AF-free survival rate of 72.83% at the 1-year follow-up in paroxysmal AF patients undergoing cryoballoon ablation. The most frequent, but reversible complication is phrenic nerve palsy with reported incidences up to 10%. All efforts must be taken to overcome this limitation, since the overall major complication rate tends to be lower in cryoballoon compared to radiofrequency ablation. In persistent AF, reported results in cryoballoon ablation had a limited success rate below 50% after a single procedure. A double balloon approach using both cryoballoon sizes might overcome some of the limitations in persistent AF. Prospective data and randomized studies are required. This article outlines the current status of cryoballoon technology in AF ablation therapy.

Molecular cloning and analysis of the human cardiac sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) gene promoter.

The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) plays a critical role in regulating Ca2+ movements in myocardium. In cardiac hypertrophy and human heart failure, the decrease in mRNA and protein levels of SERCA2 might account for the reduced diastolic Ca2+ re-uptake seen in these conditions. To investigate the regulation of human SERCA2 gene expression, an 18.6-kb human genomic clone that contains exons 1,2 and 3 of the SERCA2 gene has been isolated, and 13 kb of 5' upstream flanking sequence of which the proximal 2.5 kb of the promoter have been sequenced. Similar to the rabbit gene, the human SERCA2 promoter possesses a TATA-like box (-25 bp), a CAAT-box (-78 bp) and a number of consensus cis-regulatory elements including three Sp1 sites, a CACCC-box, and an OTF-1 binding sequence. No CArG box (present in the rabbit SERCA2 promoter) was identified in the human proximal promoter. Two putative thyroid response elements (TRE) are also present, suggesting that the human SERCA2 gene is also regulated by thyroid hormone as are the rat and rabbit genes. To study transcriptional activity of the human SERCA2 promoter in vitro, luciferase reporter plasmids containing a series of 5' deleted promoter constructs from -2577 bp to +170 bp were transfected into neonatal rat cardiomyocytes and C2C12 myotubes. The results suggest that: (a) the sequences from the transcription start site to -263 bp are necessary to obtain maximal transcriptional activity; (b) sequences from the transcription start site to -125 bp are essential for basal transcriptional activity; (c) at least one positive regulatory element is located between -263 bp and -125 bp; and (d) at least one negative regulatory element is present between -1741 bp and -412 bp.

Calcium transport proteins in the nonfailing and failing heart: gene expression and function.

In heart failure alterations of intracellular Ca2+ handling are thought to be a major reason for impaired contraction and relaxation. Peak Ca2+ transients are reduced, resting Ca2+ levels elevated, and the time course of diastolic Ca2+ decline is markedly prolonged in failing hearts. The proteins of the sarcoplasmic reticulum and the sarcolemmal Na+/Ca2+ exchanger are the most important tools for Ca2+ homeostasis in the cardiomyocyte, and their molecular cloning has allowed prediction of structure/function analysis. The investigation of function and gene expression of these proteins in failing myocardium has been an area of intensive research in recent years in order to provide a more detailed understanding of the pathophysiology of heart failure. Quantitative changes in expression of the sarcoplasmic reticulum Ca(2+)-ATPase, the ryanodine receptor, and the Na+/Ca2+ exchanger with correlations to functional alterations have been reported both in experimental animal models and in the human failing heart. However, in human heart failure these findings are currently the subject of a lively discussion because observations have apparently been in part contradictory. This review discusses the proteins involved in myocardial Ca2+ handling and describes the current state of research on expressional and functional alterations and their potential implication in the pathomechanism of heart failure.

Contribution of cAMP-phosphodiesterase inhibition and sensitization of the contractile proteins for calcium to the inotropic effect of pimobendan in the failing human myocardium.

Previous studies have shown reduced effects of cAMP-dependent positive inotropic agents in the failing human myocardium; thus other cAMP-independent mechanisms of action may be useful to increase force of contraction in this condition. The purpose of this investigation was to determine whether a positive inotropic effect of the cAMP-phosphodiesterase (PDE) inhibitor pimobendan is observed in the failing human myocardium and to study whether other factors, such as an increase in the Ca2+ sensitivity of myofilaments, play a functional role in the increase in force of contraction. Pimobendan produced a positive inotropic effect in isolated preparations from nonfailing donor hearts; however, in moderately (New York Heart Association class II-III, NYHA II-III) and severely (NYHA IV) failing myocardium, this effect was reduced. In addition, in NYHA IV specimens pimobendan inhibited the crude cAMP-PDE (crude PDE) and the isoenzymes I-III (PDE I-III) in a concentration-dependent way. As judged from the IC50 values found in this tissue for the inhibition of PDE III and of crude PDE, the potency of the compound was 18.1 times greater on PDE III. Consistent with a cAMP-PDE-dependent mechanism of action, the positive inotropic effect was potentiated by isoproterenol and inhibited by adenosine in failing myocardium. In failing myocardium, pimobendan also increased the sensitivity of skinned cardiac fibers to Ca2+ and shifted the Ca(2+)-tension relation to the left. This sensitizing effect began at 0.01 mumol/l in NYHA II-III and NYHA IV and rose to about 200% at 300 mumol/l in both groups. In contrast, the demethylated metabolite UD-CG 212 Cl failed to produce positive inotropic effects in failing myocardium alone, but in the presence of isoproterenol, it exerted an increase in force of contraction. The potency of UD-CG 212 Cl for PDE III inhibition in NYHA IV was greater than that of pimobendan. The metabolite pronouncedly decreased the sensitivity of skinned cardiac fibers to Ca2+ at 30-300 mumol/l in NYHA II-III and NYHA IV. It is concluded that in the failing human heart pimobendan inhibited PDE III and sensitized contractile proteins for Ca2+. Both effects appear to be involved in the positive inotropic effect of the compound, because its metabolite, UD-CG 212 Cl, had no effect on force of contraction and on the Ca2+ sensitivity of skinned cardiac fibers but inhibited PDE III even more potently than pimobendan.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium sensitivity and myosin light chain pattern of atrial and ventricular skinned cardiac fibers from patients with various kinds of cardiac disease.

In the present study, the Ca2(+)-sensitivity and myosin light chain patterns of skinned fibers of right atrium and left papillary muscles of 27 patients suffering from mitral valve disease (MVD, moderate heart failure), ischemic cardiomyopathy (ICM, severe heart failure), dilated cardiomyopathy (DCM, severe heart failure), and coronary heart disease (CHD, no heart failure, no atrial hypertrophy) were investigated. Myosin light chains of both chemically skinned and intact samples were studied by two-dimensional gel electrophoresis (2D-PAGE). Ca2(+)-sensitivity of ventricular fibers was about 0.14 pCa-units higher than that of atrial fibers in all groups except dilated cardiomyopathy where this difference was markedly diminished (only 0.06 pCa-units). Generally, Ca2(+)-sensitivity of skinned ventricular fibers was the same among the different heart diseases. Skinned atrial fibers from patients with dilated cardiomyopathy, however, were significantly (about 0.08 pCa-units) more sensitive for Ca2+ than those of the other groups (coronary heart disease, mitral valve disease or ischemic cardiomyopathy) which showed similar Ca2(+)-tension relationships. Ventricle-specific P-light chain forms could be observed in atrial samples from patients of all groups, whereas no atrium-specific light chain forms were detectable in any ventricular sample. It is concluded that there is no difference in Ca2(+)-sensitivity of the ventricular contractile elements of the human heart in different heart diseases. In atrial myocardium, there is an increased Ca2(+)-sensitivity of skinned fibers from hearts with dilated cardiomyopathy which is probably related to an elevation of right atrial pressure.

Inotropic response to DPI 201-106 in the failing human heart.

1. The present study was designed to characterize the positive inotropic response to DPI 201-106 in isolated papillary muscle strips obtained from heart failure patients undergoing surgery. 2. The positive inotropic responses to isoprenaline and milrinone and cardiac beta-adrenoceptor density were also determined. 3. DPI 201-106 increased the force of contraction in papillary muscle strips from patients with moderate (NYHA II-III) and severe (NYHA IV) heart failure, in a concentration-dependent manner. This positive inotropic effect was more pronounced in tissues from NYHA IV patients. Furthermore, these responses were greater than those produced by milrinone or isoprenaline. The positive inotropic effects of isoprenaline and milrinone were reduced in NYHA IV compared to NYHA II-III. Consistently, there was also a smaller density of beta-adrenoceptors in myocardium from NYHA IV than in NYHA II-III. The positive inotropic effect of Ca2+ was similar in tissues from both groups of patients. 4. The positive inotropic effect of DPI 201-106 was not antagonized by adenosine or carbachol, whereas both compounds reduced the positive inotropic effect of isoprenaline. 5. DPI 201-106 did not increase the Ca2+ -sensitivity of chemically skinned ventricular fibres, whereas a significant increase of the Ca2+ -sensitivity was obtained with trifluoperazine. 6. It is concluded that DPI 201-106 produces significant positive inotropic effects in tissue excised from the failing human heart. The lack of inhibition by adenosine and carbachol might contribute to its greater effectiveness in NYHA IV than NYHA II-III and indicates that its mechanism of action is cyclic AMP-independent. A sensitization of the contractile proteins to Ca2+ does not appear to be important for the positive inotropic action of DPI 201-106 in the failing human heart.

Myosin P-light chain isoenzymes in the human heart: evidence for diphosphorylation of the atrial P-LC form.

We studied myosin light chains (LC) of human atrium and ventricle of normal and diseased individuals by a high-resolution 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique. Atrial LCs (ALC-1, ALC-2 (= P-LC)) revealed both higher molecular weights and lower isoelectric points (IEP) than their ventricular counterparts (VLC-1, VLC-2 (= P-LC)). Different P-LC forms with their distinct myosin isoenzymes have been designated as P-LC-polymorphism and myosin P-LC isoenzymes, respectively. In the dephosphorylated state two VLC-2 forms (VLC-2 and VLC-2*) with the same MW and different IEP, but only one ALC-2 form, were found. In the partially phosphorylated state ALC-2 appeared to be single- and double-phosphorylated (three spots in the 2D-PAGE), whereas the two VLC-2 forms appeared to be single-phosphorylated each (four spots in the 2D-PAGE). Phosphoryl-transfer from ATP to the P-LC forms was studied using skinned fibers incubated with MLCK (myosin light chain kinase) and (gamma-32P)ATP. Ventricular myosin P-LC isoenzyme pattern was usually the same in normal and diseased patients: the VLC-2 to VLC-2* ratio was approx. 70/30, but in one patient with valvular heart disease (VHD) the relation was 55/45 (shift to the VLC-2* form). In hypertrophied atria of VHD patients a shift of the myosin P-LC isoenzyme pattern to the VLC-2* form occurred, too.