Myocardial Ablation of G Protein-Coupled Receptor Kinase 2 (GRK2) Decreases Ischemia/Reperfusion Injury through an Anti-Intrinsic Apoptotic Pathway

Studies from our lab have shown that decreasing myocardial G protein-coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.

Activation of the lectin pathway of complement in pig-to-human xenotransplantation models

BACKGROUND Natural IgM containing anti-Gal antibodies initiates classic pathway complement activation in xenotransplantation. However, in ischemia-reperfusion injury, IgM also induces lectin pathway activation. The present study was therefore focused on lectin pathway as well as interaction of IgM and mannose-binding lectin (MBL) in pig-to-human xenotransplantation models. METHODS Activation of the different complement pathways was assessed by cell enzyme-linked immunosorbent assay using human serum on wild-type (WT) and α-galactosyl transferase knockout (GalTKO)/hCD46-transgenic porcine aortic endothelial cells (PAEC). Colocalization of MBL/MASP2 with IgM, C3b/c, C4b/c, and C6 was investigated by immunofluorescence in vitro on PAEC and ex vivo in pig leg xenoperfusion with human blood. Influence of IgM on MBL binding to PAEC was tested using IgM depleted/repleted and anti-Gal immunoabsorbed serum. RESULTS Activation of all the three complement pathways was observed in vitro as indicated by IgM, C1q, MBL, and factor Bb deposition on WT PAEC. MBL deposition colocalized with MASP2 (Manders' coefficient [3D] r=0.93), C3b/c (r=0.84), C4b/c (r=0.86), and C6 (r=0.80). IgM colocalized with MBL (r=0.87) and MASP2 (r=0.83). Human IgM led to dose-dependently increased deposition of MBL, C3b/c, and C6 on WT PAEC. Colocalization of MBL with IgM (Pearson's coefficient [2D] rp=0.88), C3b/c (rp=0.82), C4b/c (rp=0.63), and C6 (rp=0.81) was also seen in ex vivo xenoperfusion. Significantly reduced MBL deposition and complement activation was observed on GalTKO/hCD46-PAEC. CONCLUSION Colocalization of MBL/MASP2 with IgM and complement suggests that the lectin pathway is activated by human anti-Gal IgM and may play a pathophysiologic role in pig-to-human xenotransplantation.

Electrocardiographic characteristics of patients with Ebstein's anomaly before and after ablation of an accessory atrioventricular pathway

UNLABELLED The abnormal development of the tricuspid valve in patients with Ebstein's anomaly results in several activation abnormalities including delayed intraatrial conduction, right bundle branch block (RBBB), and ventricular preexcitation. The aim of the present study was to define the ECG characteristics before and after ablation of an accessory A-V pathway (AP) in patients with Ebstein's anomaly. METHODS A series of 226 consecutive patients with Ebstein's anomaly was studied. Sixty-four patients (28%) had documented tachycardia. Thirty-three patients with recurrent tachycardia were found to have a single right-sided AP that was successfully ablated (study group). Thirty patients without tachycardia served as the control group. RESULTS Only 21 of 33 patients (62%) had a typical ECG pattern of preexcitation. In addition, none of the patients had an ECG pattern of RBBB during sinus rhythm. In contrast, 28 of 30 (93%) patients in the control group had RBBB (P < 0.001). Radiofrequency catheter ablation resulted in appearance of RBBB in 31 of 33 (94%) patients. The absence of RBBB in patients with Ebstein's anomaly and recurrent tachycardia had a 98% sensitivity and 92% specificity for the diagnosis of an AP. The positive predictive value was 91% (0.77, 0.97 CI 95%) and the negative predictive value was 98% (0.85, 0.99 CI 95%). CONCLUSION One-third of patients with Ebstein's anomaly and symptomatic tachyarrhythmias have minimal or absent ECG features of ventricular preexcitation. In these patients, the absence of RBBB pattern is a strong predictor of an AP.

Differentiating atrioventricular nodal reentrant tachycardia from tachycardia via concealed accessory pathway.

Studies analyzing the diagnostic value of 12-lead electrocardiographic criteria differentiating slow-fast atrioventricular nodal reentrant tachycardia (AVNRT) from atrioventricular reentrant tachycardia (AVRT) due to concealed accessory pathway have shown inconsistent results. In 97 patients (50 with AVNRT, 47 with AVRT) 12-lead electrocardiograms (ECGs) were recorded during sinus rhythm and tachycardia (QRS <120 ms). The ECGs were blinded for diagnosis and patient and analyzed independently by 2 electrophysiologists. The studied criteria differentiating AVNRT from AVRT included pseudo-r'/S, the presence of a retrograde P wave, RP interval, ST-segment depression >/=2 mm with the number and location of the affected leads, QRS amplitude, and cycle length alternans.

Left-septal ablation of the fast pathway in AV nodal reentrant tachycardia refractory to right septal ablation.

In more than 95% of patients with atrioventricular nodal reentrant tachycardia (AVNRT), curative treatment can be achieved with selective ablation of the slow pathway in the right-sided septum. We report a patient with typical AVNRT who had failed attempts to perform conventional right septal ablation of the slow as well as of the fast pathway and finally underwent successful ablation of the fast pathway on the left side of the interatrial septum using a transseptal approach.

Paraseptal accessory pathway in Wolff-Parkinson- White-Syndrom: ablation from the right, from the left or within the coronary sinus/middle cardiac vein?

AIMS In 1999 the consensus statement "living anatomy of the atrioventricular junctions" was published. With that new nomenclature the former posteroseptal accessory pathway (APs) are termed paraseptal APs. The aim of this study was to identify ECG features of manifest APs located in this complex paraseptal space. METHODS AND RESULTS ECG characteristics of all patients who underwent radiofrequency ablation of an AP during a 3 year period were analyzed. Of the 239 patients with one or more APs, 30 patients had a paraseptal AP with preexcitation. Compared to APs within the coronary sinus (CS) or the middle cardiac vein (MCV) the right sided paraseptal APs significantly more often showed an isoelectric delta wave in lead II and/or a negative delta wave in aVR. The left sided paraseptal APs presented a negative delta wave in II significantly more often compared to the right sided APs. CONCLUSIONS According to the site of radiofrequency ablation, paraseptal APs are classified into 4 subgroups: paraseptal right, paraseptal left, inside the CS or inside the MCV. Subtle differences in preexcitation patterns of the delta wave as well as of the QRS complex exist. However, the definitive localization of APs remains reserved to the periinterventional intracardiac electrogram analysis.

Targeting the sphingosine kinase/sphingosine 1-phosphate pathway to treat chronic inflammatory kidney diseases

Chronic kidney diseases including glomerulonephritis are often accompanied by acute or chronic inflammation that leads to an increase in extracellular matrix (ECM) production and subsequent glomerulosclerosis. Glomerulonephritis is one of the leading causes for end-stage renal failure with high morbidity and mortality, and there are still only a limited number of drugs for treatment available. In this MiniReview, we discuss the possibility of targeting sphingolipids, specifically the sphingosine kinase 1 (SphK1) and sphingosine 1-phosphate (S1P) pathway, as new therapeutic strategy for the treatment of glomerulonephritis, as this pathway was demonstrated to be dysregulated under disease conditions. Sphingosine 1-phosphate is a multifunctional signalling molecule, which was shown to influence several hallmarks of glomerulonephritis including mesangial cell proliferation, renal inflammation and fibrosis. Most importantly, the site of action of S1P determines the final effect on disease progression. Concerning renal fibrosis, extracellular S1P acts pro-fibrotic via activation of cell surface S1P receptors, whereas intracellular S1P was shown to attenuate the fibrotic response. Interference with S1P signalling by treatment with FTY720, an S1P receptor modulator, resulted in beneficial effects in various animal models of chronic kidney diseases. Also, sonepcizumab, a monoclonal anti-S1P antibody that neutralizes extracellular S1P, and a S1P-degrading recombinant S1P lyase are promising new strategies for the treatment of glomerulonephritis. In summary, especially due to the bifunctionality of S1P, the SphK1/S1P pathway provides multiple target sites for the treatment of chronic kidney diseases.

A phosphoinositide 3-kinase (PI3K)-serum- and glucocorticoid-inducible kinase 1 (SGK1) pathway promotes Kv7.1 channel surface expression by inhibiting Nedd4-2 protein

Epithelial cell polarization involves several kinase signaling cascades that eventually divide the surface membrane into an apical and a basolateral part. One kinase, which is activated during the polarization process, is phosphoinositide 3-kinase (PI3K). In MDCK cells, the basolateral potassium channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Here, we demonstrate that Kv7.1 surface expression requires tonic PI3K activity as PI3K inhibition triggers endocytosis of these channels in polarized MDCK. Pharmacological inhibition of SGK1 gave similar results as PI3K inhibition, whereas overexpression of constitutively active SGK1 overruled it, suggesting that SGK1 is the primary downstream target of PI3K in this process. Furthermore, knockdown of the ubiquitin ligase Nedd4-2 overruled PI3K inhibition, whereas a Nedd4-2 interaction-deficient Kv7.1 mutant was resistant to both PI3K and SGK1 inhibition. Altogether, these data suggest that a PI3K-SGK1 pathway stabilizes Kv7.1 surface expression by inhibiting Nedd4-2-dependent endocytosis and thereby demonstrates that Nedd4-2 is a key regulator of Kv7.1 localization and turnover in epithelial cells.

Status and Causal Pathway Assessments Supporting River Basin Management

The European Water Framework Directive (WFD) requires a status assessment of all water bodies. If that status is deteriorated, the WFD urges the identification of its potential causes in order to be able to suggest appropriate management measures. The instrument of investigative monitoring allows for such identification, provided that appropriate tools are available to link the observed effects to causative stressors, while unravelling confounding factors. In this chapter, the state of the art of status and causal pathway assessment is described for the major stressors responsible for the deterioration of European water bodies, i.e. toxicity, acidification, salinisation, eutrophication and oxygen depletion, parasites and pathogens, invasive alien species, hydromorphological degradation, changing water levels as well as sediments and suspended matter. For each stressor, an extensive description of the potential effects on the ecological status is given. Secondly, stressor-specific abiotic and biotic indicators are described that allow for a first indication of probable causes, based on the assessment of available monitoring data. Subsequently, more advanced tools for site-specific confirmation of stressors at hand are discussed. Finally, the local status assessments are put into the perspective of the risk for downstream stretches in order to be able to prioritise stressors and to be able to select appropriate measures for mitigation of the risks resulting from these stressors.

White matter pathway organization of the reward system is related to positive and negative symptoms in schizophrenia

The reward systemin schizophrenia has been linked to the emergence of delusions on the one hand and to negative symptoms such as affective flattening on the other hand. Previous Diffusion Tensor Imaging (DTI) studies reported white matter microstructure alterations of regions related to the reward system. The present study aimed at extending these findings by specifically investigating connection pathways of the reward system in schizophrenia. Therefore, 24 patients with schizophrenia and 22 healthy controls matched for age and gender underwent DTI-scans. Using a probabilistic fiber tracking approachwe bilaterally extracted pathways connecting the ventral tegmental area (VTA) with the nucleus accumbens (NAcc), themedial and lateral orbitofrontal cortices (mOFC, lOFC), the dorsolateral prefrontal cortex (dlPFC) and the amygdala; as well as pathways connecting NAcc with mOFC, lOFC, dlPFC and amygdala resulting in a total of 18 connections. Probability indices forming part of a bundle of interest (PIBI) were compared between groups using independent t-tests. In 6 connection pathways PIBI-valueswere increased in schizophrenia. In 3 of these pathways the spatial extension of connection pathways was decreased. In schizophrenia patients, there was a negative correlation of PIBI-values and PANSS negative scores in the left VTA–amygdala and in the left NAcc–mOFC connection. A sum score of delusions and hallucinations correlated positively with PIBI-values of the left amygdala–NAcc connection. Structural organization of specific segments ofwhite matter pathways of the reward systemin schizophrenia may contribute to the emergence of delusions and negative symptoms in schizophrenia.

The 17, 20-lyase activity of cytochrome p450c17 from human fetal testis favors the delta5 steroidogenic pathway.

Cytochrome P450c17 catalyzes both 17alpha-hydroxylation and 17,20-lyase conversion of 21-carbon steroids to 19-carbon precursors of sex steroids. P450c17 can mediate testosterone biosynthesis via the conversion of pregnenolone to dehydroepiandrosterone (the delta(5) pathway) or via conversion of progesterone to androstenedione (the delta(4) pathway). In many species, the 17, 20-lyase activity of P450c17 for one pathway dominates, reflecting the preferred steroidogenic pathway of that species. All studies of recombinant human P450c17 and of human adrenal microsomes have found high 17, 20-lyase activity only in the delta(5) pathway. Because the 17, 20-lyase activities in both the delta(4) and delta(5) pathways for testicular P450c17 have not been directly compared, however, it is not known if the delta(5) pathway dominates in the human testis. To resolve this issue, we assayed the conversion of 17alpha-hydroxypregnenolone to dehydroepiandrosterone (delta(5) 17, 20-lyase activity) and of 17alpha-hydroxyprogesterone to androstenedione (delta(4) 17, 20-lyase activity) by human fetal testicular microsomes. We obtained apparent Michaelis constant (K(m)) and maximum velocity (V(max)) values of 1.0 microM and 0.73 pmol.min(-1). microg(-1) for delta(5) 17, 20-lyase activity and of 3.5 microM and 0.23 pmol.min(-1). microg(-1) for delta(4) 17, 20-lyase activity. Catalytic efficiencies, expressed as the ratio V(max)/K(m), were 0.73 and 0.066 for the delta(5) and delta(4) reactions, respectively, indicating 11-fold higher preference for the delta(5) pathway. We conclude that the majority of testosterone biosynthesis in the human testis proceeds through the conversion of pregnenolone to dehydroepiandrosterone via the delta(5) pathway.