Universitätsbibliothek

Aims Inhibition of neprilysin and angiotensin II receptor by sacubitril/valsartan (Val) (LCZ696) reduces mortality in heart failure (HF) patients compared with sole inhibition of renin–angiotensin system. Beneficial effects of increased natriuretic peptide levels upon neprilysin inhibition have been proposed, whereas direct effects of sacubitrilat (Sac) (LBQ657) on myocardial Ca\(^{2+}\) cycling remain elusive. Methods and results Confocal microscopy (Fluo‐4 AM) was used to investigate pro‐arrhythmogenic sarcoplasmic reticulum (SR) Ca\(^{2+}\) leak in freshly isolated murine and human ventricular cardiomyocytes (CMs) upon Sac (40 μmol/L)/Val (13 μmol/L) treatment. The concentrations of Sac and Val equalled plasma concentrations of LCZ696 treatment used in PARADIGM‐HF trial. Epifluorescence microscopy measurements (Fura‐2 AM) were performed to investigate effects on systolic Ca\(^{2+}\) release, SR Ca\(^{2+}\) load, and Ca\(^{2+}\)‐transient kinetics in freshly isolated murine ventricular CMs. The impact of Sac on myocardial contractility was evaluated using in toto‐isolated, isometrically twitching ventricular trabeculae from human hearts with end‐stage HF. Under basal conditions, the combination of Sac/Val did not influence diastolic Ca\(^{2+}\)‐spark frequency (CaSpF) nor pro‐arrhythmogenic SR Ca\(^{2}\) leak in isolated murine ventricular CMs (n CMs/hearts = 80/7 vs. 100/7, P = 0.91/0.99). In contrast, Sac/Val treatment reduced CaSpF by 35 ± 9% and SR Ca\(^{2+}\) leak by 45 ± 9% in CMs put under catecholaminergic stress (isoproterenol 30 nmol/L, n = 81/7 vs. 62/7, P < 0.001 each). This could be attributed to Sac, as sole Sac treatment also reduced both parameters by similar degrees (reduction of CaSpF by 57 ± 7% and SR Ca2+ leak by 76 ± 5%; n = 101/4 vs. 108/4, P < 0.01 each), whereas sole Val treatment did not. Systolic Ca2+ release, SR Ca\(^{2+}\) load, and Ca\(^{2+}\)‐transient kinetics including SERCA activity (k\(_{SERCA}\)) were not compromised by Sac in isolated murine CMs (n = 41/6 vs. 39/6). Importantly, the combination of Sac/Val and Sac alone also reduced diastolic CaSpF and SR Ca\(^{2+}\) leak (reduction by 74 ± 7%) in human left ventricular CMs from patients with end‐stage HF (n = 71/8 vs. 78/8, P < 0.05 each). Myocardial contractility of human ventricular trabeculae was not acutely affected by Sac treatment as the developed force remained unchanged over a time course of 30 min (n trabeculae/hearts = 3/3 vs. 4/3). Conclusion This study demonstrates that neprilysin inhibitor Sac directly improves Ca\(^{2+}\) homeostasis in human end‐stage HF by reducing pro‐arrhythmogenic SR Ca\(^{2+}\) leak without acutely affecting systolic Ca\(^{2+}\) release and inotropy. These effects might contribute to the mortality benefits observed in the PARADIGM‐HF trial.

Osmotic stimulus or stress results in vasopressin release. Animal and human in vitro studies have shown that inflammatory parameters, such as interle ukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha), increase in parallel in the central nervous system and bronchial, corneal or intestinal epithelial cell lines in response to osmotic stimulus. Whether osmotic stimulus directly causes a systemic inflammatory response in humans is unknown. We therefore investigated the influence of osmotic stimulus on circulatory markers of systemic inflammation in healthy volunteers. In this prospective cohort study, 44 healthy volunteers underwent a standardized test protocol with an osmotic stimulus leading into the hyperosmotic/hypernatremic range (serum sodium >= 150 mmol/L) by hypertonic saline infusion. Copeptin - a marker indicating vasopressin activity - serum sodium and osmolality, plasma IL-8 and TNF-alpha were measured at baseline and directly after osmotic stimulus. Median (range) serum sodium increased from 141 mmol/L (136, 147) to 151 mmol/L (145, 154) (P < 0.01), serum osmolality increased from 295 mmol/L (281, 306) to 315 mmol/L (304, 325) (P < 0.01). Median (range) copeptin increased from 4.3 pg/L (1.1, 21.4) to 28.8 pg/L (19.9, 43.4) (P < 0.01). Median (range) IL-8 levels showed a trend to decrease from 0.79 pg/mL (0.37, 1.6) to 0.7 pg/mL (0.4, 1.9) (P < 0.09) and TNF-alpha levels decreased from 0.53 pg/mL (0.11, 1.1) to 0.45 pg/mL (0.1 2, 0.97) (P < 0.036). Contrary to data obtained in vitro, circulating proinflammatory cytokines tend to or decrease in human plasma after osmotic stimulus. In this study, osmotic stimulus does not increase circulating markers of systemic inflammation.

Das Herz ist physiologisch auf einen fein regulierten und ausgeglichenen bioenergetischen Energiehaushalt angewiesen, um auf akute Belastungssituationen adäquat reagieren zu können und oxidativen Stress zu vermeiden. Ca2+ reguliert zentral sowohl die zyklischen Kontraktions-/Relaxationsprozesse (ECC) als auch unmittelbar den mitochondrialen Metabolismus. Der ECC liegt in den Kardiomyozyten die Ca2+- Freisetzung durch die RyR2 zu Grunde; die IP3 Rezeptoren des sarkoplasmatischen Retikulums (SR) führen davon unabhängig zu einer Ca2+ Freisetzung aus dem SR. Diese IP3R vermittelten Signale werden in den räumlich nahe gelegenen Mitochondrien zum Teil über den mRyR1 in die mitochondriale Matrix aufgenommen und stimulieren dort langfristig die oxidative Phosphorylierung und den Erhalt der antioxidativen Kapazität. Die enge räumliche Nähe zwischen SR und Mitochondrien wird durch Strukturproteine wie Mitofusin 2 (Mfn2) ergänzt, die das SR mit der äußeren Mitochondrienmembran koppeln und so die Ca2+-Interaktion beeinflussen. Ziel der Arbeit war, den Effekt von Mfn2 Defizienz auf die IP3 induzierte mitochondriale Ca2+-Regulation in Kardiomyozyten zu evaluieren. Dazu erfolgten Fluoreszenzfärbungen an adulten isolierten Ventrikelkardiomyozyten kardiospezifischer Mfn2 Knock-Out (KO) Mäusen bzw. deren wildtypischen Geschwistertieren (WT). Erhobene Parameter umfassten das mitochondriale Ca2+, das mitochondriale Membranpotenzial, die mitochondriale Superoxidbildung und mitochondriale ATP-Gehalt. Die Ergebnisse bestätigten eine Signalachse, bei der die Stimulation von isolierten murinen Kardiomyozyten mit dem IP3 Agonisten ET-1 zu einer mitochondrialen Ca2+ Aufnahme führte, dem Erhalt des mitochondrialen Membranpotenzials diente und der ATP Gehalt stiegt. Bei induzierter kardiospezifischer Ablation von Mfn2 geht diese SR-mitochondriale Interaktion verloren, und es entstand ein energetisches Defizit sowie eine verminderte Superoxidbildung. Bei beta-adrenerger Stimulation mit Isoproterenol (ISO) resultierte in WT zwar eine mitochondriale Ca2+-Aufnahme, allerdings ein Abfall des ATP-Gehaltes. In den Mfn2 defizienten Kardiomyozyten zeigte sich eine Steigerung des ATP-Gehaltes auch auf beta-adrenerge Stimulation, die einen energetischen Kompensationsmechanismus in den Mfn2 KO Tieren vermuten lässt. Dies identifiziert Mfn2 als kritische Strukturkomponente für die basale bioenergetische Adaptation der durch IP3R-mRyR1 vermittelten Signalachse unter physiologischen Bedingungen.

A liquid chromatography tandem mass spectrometry method for the analysis of ten kinase inhibitors (afatinib, axitinib, bosutinib,cabozantinib, dabrafenib, lenvatinib, nilotinib, osimertinib, ruxolitinib, and trametinib) in human serum and plasma for theapplication in daily clinical routine has been developed and validated according to the US Food and Drug Administration andEuropean Medicines Agency validation guidelines for bioanalytical methods. After protein precipitation of plasma samples withacetonitrile, chromatographic separation was performed at ambient temperature using a Waters XBridge® Phenyl 3.5μm(2.1×50 mm) column. The mobile phases consisted of water-methanol (9:1, v/v) with 10 mM ammonium bicarbonate as phase A andmethanol-water (9:1, v/v) with 10 mM ammonium bicarbonate as phase B. Gradient elution was applied at a flow rate of 400μL/min. Analytes were detected and quantified using multiple reaction monitoring in electrospray ionization positive mode. Stableisotopically labeled compounds of each kinase inhibitor were used as internal standards. The acquisition time was 7.0 min perrun. All analytes and internal standards eluted within 3.0 min. The calibration curves were linear over the range of 2–500 ng/mLfor afatinib, axitinib, bosutinib, lenvatinib, ruxolitinib, and trametinib, and 6–1500 ng/mL for cabozantinib, dabrafenib, nilotinib,and osimertinib (coefficients of correlation≥0.99). Validation assays for accuracy and precision, matrix effect, recovery,carryover, and stability were appropriate according to regulatory agencies. The rapid and sensitive assay ensures high throughputand was successfully applied to monitor concentrations of kinase inhibitors in patients.

Inherited cardiomyopathies are characterized by clinical and genetic heterogeneity that challenge genetic diagnostics. In this study, we examined the diagnostic benefit of exome data compared to targeted gene panel analyses, and we propose new candidate genes. We performed exome sequencing in a cohort of 61 consecutive patients with a diagnosis of cardiomyopathy or primary arrhythmia, and we analyzed the data following a stepwise approach. Overall, in 64% of patients, a variant of interest (VOI) was detected. The detection rate in the main sub-cohort consisting of patients with dilated cardiomyopathy (DCM) was much higher than previously reported (25/36; 69%). The majority of VOIs were found in disease-specific panels, while a further analysis of an extended panel and exome data led to an additional diagnostic yield of 13% and 5%, respectively. Exome data analysis also detected variants in candidate genes whose functional profile suggested a probable pathogenetic role, the strongest candidate being a truncating variant in STK38. In conclusion, although the diagnostic yield of gene panels is acceptable for routine diagnostics, the genetic heterogeneity of cardiomyopathies and the presence of still-unknown causes favor exome sequencing, which enables the detection of interesting phenotype–genotype correlations, as well as the identification of novel candidate genes.