Einfluss von Phosphodiesterase 5-Inhibitoren auf die Escherichia coli Hämolysin und Lipopolysaccharid vermittelte Kardiodepression am isolierten Rattenherzen

Durch Mikroorganismen, wie Bakterien, Viren oder Pilze kommt es zu Primärinfektionen, in deren Folge sich eine Sepsis entwickeln kann. Die häufigsten Sepsisverursacher sind Bakterien, wobei der Tatsache, dass das Krankheitsbild der Sepsis nicht an die Anwesenheit intakter Bakterien in der Zirkulation gebunden ist sondern die hämatogene Streuung sezernierter Exotoxine und endotoxinhaltiger Zellfragmente zur Induktion einer Sepsis ausreichen, besondere Bedeutung beigemessen werden muss. Die während einer Sepsis auftretenden kardiovaskulären Veränderungen tragen entscheidend zur Induktion des septischen Multiorganversagens sowie des septischen Schocks bei. Es ist dem Herzen im Zuge einer akuten septischen Kardiomyopathie häufig nicht möglich, seine Pumpleistung so zu erhöhen, wie es zur Aufrechterhaltung eines adäquaten Blutdrucks bei stark erniedrigtem systemischem Widerstand nötig wäre. Die septische Kardiomyopathie kann durch die Wirkung kardiodepressiver Zytokine wie TNF-alpha und Il-1beta hervorgerufen werden; daneben sind Mikrozirkula-tionsstörungen innerhalb des Myokards, welche auch ohne globale Minderperfusion zu einer hypoxisch bedingten Pumpinsuffizienz führen können, als mögliche Ursache anzusehen. Zyklische Nukleotide stellen dabei einen wichtigen Bestandteil in der Regulation von koronarer Vasomotorik und kardialer Kontraktilität dar, überdies spielen sie auch eine Rolle in der Pathophysiologie der Sepsis. Die vorliegende Arbeit behandelt die Frage, ob Phosphodiesterase (PDE5) -Inhibitoren den kardiodepressiven Effekt gram-negativer Bakterien respektive deren Endo- und Exotoxinen zumindest teilweise aufheben und so zu einem günstigeren Verlauf der Sepsis beitragen können. Dazu wurden isolierte Rattenherzen mit Escherichia coli Hämolysin (ECH) und Lipopolysaccharid (LPS) perfundiert. Die dadurch generierte Kardiodepression sowie Vasokonstriktion im Koronargefäßsystem sollten mit Hilfe von Sildenafil und Zaprinast inhibiert werden, was partiell gelang. Desweiteren wurde durch die Aufarbeitung von Ventrikelpräparaten eine verminderte Freisetzung kardio-depressiver Mediatoren, insbesondere von TNF-alpha und Cysteinyl-Leukotrienen (Cys-LT), nachgewiesen. Eine Beeinflussung der Genexpression hingegen konnte nicht gezeigt werden. Für die LPS-Versuche stellt sich der protektive Effekt folgendermaßen dar: Der durch die PDE5-Inhibitoren erhöhte cGMP-Spiegel führt zu einer koronaren Vasodilatation, welche Mikrozirkulationsstörungen vorbeugt, was zur Verbesserung der Myokardversorgung und einer verminderten Freisetzung von TNF-alpha führt. Durch diese zeitige Intervention in die teilweise selbstunterhaltenden Mechanismen können der TNF-alpha abhängige kardiodepressive Effekt, aber auch die Aktivierung der cNOS/iNOS, des Sphingomyelinasesignalwegs, der freien Radikalliberation und der Apoptose antagonisiert werden. Im Falle der ECH-Versuche steht ebenfalls die vasodilatative Wirkung der PDE5-Inhibitoren im Koronargefäßsystem im Vordergrund. Diese beruht sowohl auf einer prolongierten cGMP Wirkung als auch auf einer herabgesetzten Cys-LT Liberation. Die verbesserte Perfusion verhindert die sich selbstunterhaltende, ischämisch bedingte Cys-LT Freisetzung und verringert so eine weitere Erhöhung des koronaren Gefäßwiderstandes. Infolge der Senkung des koronaren Gefäßwiderstandes wird die Kardiodepression nahezu vollständig aufgehoben. Die gewonnenen Ergebnisse deuten auf ein protektives Potential der PDE5-Inhibitoren bezüglich der septischen Kardiomyopathie hin.Microorganisms like bacteria, viruses and fungi cause primary infections, which can lead to sepsis. In fact, sepsis is primarily caused by bacteria. Induction of septicemia is not dependent on the presence of intact bacterial organisms in the blood circulation. Hematogenic spreading of secreted bacterial exotoxins as well as cellular fragments containing endotoxins can also lead to sepsis. Cardiovascular alterations due to sepsis crucially contribute to multiple organ dysfunction syndrome and to septic shock. The heart of septic patients is frequently unable to increase cardiac output adequately to compensate the systemic fall in blood pressure. On the one hand septic cardiomyopathy can be induced by cardiodepressive cytokines like TNF-alpha or Il-1beta and on the other hand by microcirculatory dysfunctions within the myocardium, which even without global ischemia may lead to a decreased cardiac output due to hypoxia. Cyclic nucleotides play an important role in coronary vasoconstriction and cardial depression and also in pathophysiology of sepsis. The present study investigates in how far the phosphodiesterase (PDE5) inhibitors Sildenafil and Zaprinast could be able to inhibit the cardiodepressive effect of gram–negative bacteria and their endo- and exotoxins respectively, which then could lead to an improvement of the prognosis. Isolated rat hearts were perfused with Escherichia coli hemolysin (ECH) and lipopolysaccharides (LPS) respectively which lead to cardiodepression and coronary vasoconstriction. After treatment with Sildenafil and Zaprinast respectively this effect could be partially decreased. Analysis of samples gained from ventricular tissue revealed a decreased liberation of cardiodepressive mediators, such as TNF-alpha and cysteinyl-leukotrienes (Cys-LT). Alterations in gene expression could not be shown. The protective effect of the PDE5-inhibitors in the LPS perfused rat hearts can be explained in the following way: PDE5-inhibitor elevate the cGMP levels which lead to coronary vasodilatation inhibiting microcirculatory dysfunction. This improves myocardial supply and decreases TNF-alpha liberation. The early intervention in the partially self engaging mechanisms inhibit the TNF-alpha dependent cardiodepressive effect, activation of the cNOS/iNOS in the sphingomyelinase signaling pathway, free radical liberation and apoptosis. The main effect of PDE5-inhibotor treatment on ECH perfused hearts is a coronary vasodilatation due to a prolonged cGMP impact and a decreased Cys-LT liberation. PDE5-inhibitors decrease self engaging, ischemic induced Cys-LT liberation, which in the end leads to a minimized increase in coronary vascular resistance. Due to the decrease of the coronary resistance cardiodepression can be abolished nearly completely. All results gained in this study recommend a beneficial potential of PDE5-inhibitors in septic cardiomyopathy

Phosphoproteomic characterization of the signaling network resulting from activation of chemokine receptor CCR2

Leukocyte recruitment is a universal feature of tissue inflammation and regulated by the interactions of chemokines with their G protein-coupled receptors (GPCRs). Activation of CC chemokine receptor 2 (CCR2) by its cognate chemokine ligands, including CC chemokine ligand 2 (CCL2), plays a central role in recruitment of monocytes in several inflammatory diseases. In this study, we used phosphoproteomics to conduct an unbiased characterization of the signaling network resulting from CCL2 activation of CCR2. Using data-independent acquisition (DIA) MS analysis, we quantified both the proteome and phosphoproteome in FlpIn-HEK293T cells stably expressing CCR2 at six time points after activation with CCL2. Differential expression analysis identified 699 significantly regulated phosphorylation sites on 441 proteins. As expected, many of these proteins are known to participate in canonical signal transduction pathways and in the regulation of actin cytoskeleton dynamics, including numerous guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Moreover, we identified regulated phosphorylation sites in numerous proteins that function in the nucleus, including several constituents of the nuclear pore complex. The results of this study provide an unprecedented level of detail of CCR2 signaling and identify potential targets for regulation of CCR2 function

Ubr1-induced selective endophagy/autophagy protects against the endosomal and Ca2+-induced proteostasis disease stress

The cellular defense mechanisms against cumulative endo-lysosomal stress remain incompletely understood. Here, we iden tify Ubr1 as a protein quality control (QC) E3 ubiquitin-ligase that counteracts proteostasis stresses by facilitating endosomal cargo-selective autophagy for lysosomal degradation. Astrocyte regulatory cluster membrane protein MLC1 mutations cause endosomal compartment stress by fusion and enlargement. Partial lysosomal clearance of mutant endosomal MLC1 is accomplished by the endosomal QC ubiquitin ligases, CHIP and Ubr1 via ESCRT-dependent route. As a consequence of the endosomal stress, a supportive QC mechanism, dependent on both Ubr1 and SQSTM1/p62 activities, targets ubiquit inated and arginylated MLC1 mutants for selective endosomal autophagy (endophagy). This QC pathway is also activated for arginylated Ubr1-SQSTM1/p62 autophagy cargoes during cytosolic Ca2+-assault. Conversely, the loss of Ubr1 and/or arginylation elicited endosomal compartment stress. These fndings underscore the critical housekeeping role of Ubr1 and arginylation-dependent endophagy/autophagy during endo-lysosomal proteostasis perturbations and suggest a link of Ubr1 to Ca2+ homeostasis and proteins implicated in various diseases including cancers and brain disorder

Ligand-dependent spatiotemporal signaling profiles of the mu-opioid receptor are controlled by distinct protein-interaction networks

Ligand-dependent differences in the regulation and internalization of the mu-opioid receptor (MOR) have been linked to the severity of adverse effects that limit opiate use in pain management. MOR activation by morphine or [D-Ala2,N-MePhe4,Gly-ol]-enkephalin (DAMGO) causes differences in spatiotemporal signaling dependent on MOR distribution at the plasma membrane. Morphine stimulation of MOR activates a Gai/o–Gbg–protein kinase C (PKC)a phosphorylation pathway that limits MOR distribution and is associated with a sustained increase in cytosolic extracellular signal–regulated kinase (ERK) activity. In contrast, DAMGO causes a redistribution of the MOR at the plasma membrane (before receptor internalization), that facilitates transient activation of cytosolic and nuclear ERK. Here, we used proximity biotinylation proteomics to dissect the different protein-interaction networks that underlie the spatiotemporal signaling of morphine and DAMGO. We found that DAMGO, but not morphine, activates Ras‐related C3 botulinum toxin substrate 1 (Rac1). Both Rac1 and nuclear ERK activity was dependent on the scaffolding proteins IQ motif–containing GTPase-activating protein-1 (IQGAP1) and Crk-like protein (CRKL). In contrast, morphine increased the proximity of the MOR to desmosomal proteins, which form specialized and highly ordered membrane domains. Knockdown of two desmosomal proteins, junction plakoglobin (JUP) or desmocolin-1 (DSC1), switched the morphine spatiotemporal signaling profile to mimic that of DAMGO, resulting in a transient increase in nuclear ERK activity. The identification of the MOR-interaction networks that control differential spatiotemporal signaling reported here is an important step towards understanding how signal compartmentalization contributes to opioid-induced responses including anti-nociception and the development of tolerance and dependence

Methylome and proteome integration in human skeletal muscle uncover group and individual responses to high-intensity interval training.

Exercise is a major beneficial contributor to muscle metabolism, and health benefits acquired by exercise are a result of molecular shifts occurring across multiple molecular layers (i.e., epigenome, transcriptome, and proteome). Identifying robust, across-molecular level targets associated with exercise response, at both group and individual levels, is paramount to develop health guidelines and targeted health interventions. Sixteen, apparently healthy, moderately trained (VO2 max = 51.0 ± 10.6 mL min-1  kg-1 ) males (age range = 18-45 years) from the Gene SMART (Skeletal Muscle Adaptive Responses to Training) study completed a longitudinal study composed of 12-week high-intensity interval training (HIIT) intervention. Vastus lateralis muscle biopsies were collected at baseline and after 4, 8, and 12 weeks of HIIT. DNA methylation (~850 CpG sites) and proteomic (~3000 proteins) analyses were conducted at all time points. Mixed models were applied to estimate group and individual changes, and methylome and proteome integration was conducted using a holistic multilevel approach with the mixOmics package. A total of 461 proteins significantly changed over time (at 4, 8, and 12 weeks), whilst methylome overall shifted with training only one differentially methylated position (DMP) was significant (adj.p-value 0.5, among them are two novel exercise-related proteins, LYRM7 and EPN1. Integration analysis showed bidirectional relationships between the methylome and proteome. We showed a significant influence of HIIT on the epigenome and more so on the proteome in human muscle, and uncovered groups of proteins clustering according to similar patterns across the exercise intervention. Individual responses to exercise were observed in the proteome with novel mitochondrial and metabolic proteins consistently changed across individuals. Future work is required to elucidate the role of these proteins in response to exercise

Modularity and hormone sensitivity of the Drosophila melanogaster insulin receptor/target of rapamycin interaction proteome

First systematic analysis of the evolutionary conserved InR/TOR pathway interaction proteome in Drosophila.Quantitative mass spectrometry revealed that 22% of identified protein interactions are regulated by the growth hormone insulin affecting membrane proximal as well as intracellular signaling complexes.Systematic RNA interference linked a significant fraction of network components to the control of dTOR kinase activity.Combined biochemical and genetic data suggest dTTT, a dTOR-containing complex required for cell growth control by dTORC1 and dTORC2 in vivo

Skeletal muscle NOX4 is required for adaptive responses that prevent insulin resistance

Reactive oxygen species (ROS) generated during exercise are considered integral for the health-promoting effects of exercise. However, the precise mechanisms by which exercise and ROS promote metabolic health remain unclear. Here, we demonstrate that skeletal muscle NADPH oxidase 4 (NOX4), which is induced after exercise, facilitates ROS-mediated adaptive responses that promote muscle function, maintain redox balance, and prevent the development of insulin resistance. Conversely, reductions in skeletal muscle NOX4 in aging and obesity contribute to the development of insulin resistance. NOX4 deletion in skeletal muscle compromised exercise capacity and antioxidant defense and promoted oxidative stress and insulin resistance in aging and obesity. The abrogated adaptive mechanisms, oxidative stress, and insulin resistance could be corrected by deleting the H2O2-detoxifying enzyme GPX-1 or by treating mice with an agonist of NFE2L2, the master regulator of antioxidant defense. These findings causally link NOX4-derived ROS in skeletal muscle with adaptive responses that promote muscle function and insulin sensitivity

Exploring the selectivity and engineering potential of an NRPS condensation domain involved in the biosynthesis of the thermophilic siderophore fuscachelin

In nonribosomal peptide synthesis, condensation (C) domains are key catalytic domains that most commonly link carrier protein bound substrates to form peptides or depsipeptides. While adenylation domains have been well characterized due to their role in the selection of monomers and hence as gate keepers in nonribosomal peptide biosynthesis, C-domains have been the subject of debate as they do not have apparent “A-domain like” side chain selectivity for their acceptor substrates. To probe the selectivity and specificity of C-domains, here we report our biochemical and structural characterization of the C3-domain from the biosynthesis of the siderophore fusachelin. Our results show that this C-domain is not broadly flexible for monomers bearing significantly alternated side chains or backbones, which suggests there can be a need to consider C-domain specificity for acceptor substrates when undertaking NRPS engineering

Restriction of essential amino acids dictates the systemic metabolic response to dietary protein dilution

Dietary protein dilution (DPD) promotes metabolic-remodelling and -health but the precise nutritional components driving this response remain elusive. Here, by mimicking amino acid (AA) supply from a casein-based diet, we demonstrate that restriction of dietary essential AA (EAA), but not non-EAA, drives the systemic metabolic response to total AA deprivation; independent from dietary carbohydrate supply. Furthermore, systemic deprivation of threonine and tryptophan, independent of total AA supply, are both adequate and necessary to confer the systemic metabolic response to both diet, and genetic AA-transport loss, driven AA restriction. Dietary threonine restriction (DTR) retards the development of obesity-associated metabolic dysfunction. Liver-derived fibroblast growth factor 21 is required for the metabolic remodelling with DTR. Strikingly, hepatocyte-selective establishment of threonine biosynthetic capacity reverses the systemic metabolic response to DTR. Taken together, our studies of mice demonstrate that the restriction of EAA are sufficient and necessary to confer the systemic metabolic effects of DPD.These studies were supported by Monash Biomedicine Discovery Institute laboratory start-up funds to A.J.R. as well as a Monash Joint Science-Medicine Interdisciplinary Research Seed Funding to A.J.R. and M.D.W.P

Restriction of essential amino acids dictates the systemic metabolic response to dietary protein dilution

Dietary protein dilution, where protein is reduced and replaced by other nutrient sources without caloric restriction, promotes metabolic health via the hepatokine Fgf21. Here, the authors show that essential amino acids threonine and tryptophan are necessary and sufficient to induce these effects