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

Micro-solid oxide fuel cells running on reformed hydrocarbon fuels

Micro‐solid oxide fuel cell (micro‐SOFC) systems are predicted to have a high energy density and specific energy and are potential power sources for portable electronic devices. A micro‐SOFC system is under development in the frame of the ONEBAT project [1‐3]. In this presentation, we report on the fabrication and characterization of a sub‐system assembly consisting of a startup heater and a micro‐reformer bonded to a Si chip with electrochemically‐active micro‐SOFC membranes. A functional carrier including fluidic channels for gas feed and integrated heaters was bonded to a microreformer with an overall size of 12.7 mm x 12.7 mm x 1.9 mm [4‐7]. As a catalyst, a foam‐like material made of ceria‐zirconia nanoparticles doped with rhodium was used to fill the 58.5 mm3 reformer cavity. This micro‐reformer allows for high methane and butane conversion of > 90 % with a hydrogen selectivity of > 80 % at 550 °C in the reformer [7, 8]. A silicon chip with 30 free‐standing micro‐SOFC membranes (390 μm x 390 μm) with a thickness of less than 500 nm was bonded to the carrier‐reformer assembly described above. The micro‐SOFC membrane consisted of an yttria‐ stabilized zirconia thin film electrolyte. Both Pt‐based and ceramic‐based electrode materials were tested regarding the thermal stability and carbon poisoning at temperatures below 600 °C. The functional‐carrier mirco‐reformer micro‐SOFC assembly was electrochemically tested with hydrocarbon fuel between 300 °C and 600 °C. The fuel cell performance and the microstructural evolution of the anode are discussed as well

A thermally self-sustained micro-power plant with integrated micro-solid oxide fuel cells, micro-reformer and functional micro-fluidic carrier

Low temperature micro-solid oxide fuel cell (micro-SOFC) systems are an attractive alternative power source for small-size portable electronic devices due to their high energy efficiency and density. Here, we report a thermally self-sustainable reformer – micro-SOFC assembly. The device consists of a micro-reformer bonded to a silicon chip containing 30 micro-SOFC membranes and a functional glass carrier with gas channels and screen-printed heaters for start-up. Thermal independence of the device from the externally powered heater is achieved by this exothermic reforming reaction above 470 °C. The reforming reaction and the fuel gas flow rate of the n-butane/air gas mixture controls the operation temperature and gas composition on the micro-SOFC membrane. In the temperature range between 505 °C and 570 °C, the gas composition after the micro-reformer consists of 12 vol% to 28 vol% H2. An open-circuit voltage of 1.0 V and maximum power density of 47 mW/cm2 at 565 °C is achieved with the on-chip produced hydrogen at the micro-SOFC membranes

Not1 reduction decreases 4E-BP mRNA through the promoter region of 4E-BP gene.

<p><b>A</b>. Schematic of reporter constructs in which the promoter region, 5’UTR and/or 3’UTR were swapped with the counterpart(s) of 4E-BP gene. (see 3B). <b>B</b>. Changes of the reporter mRNA levels by Not1 depletion. Cells were subjected to Not1 RNAi and transfection with same amounts of reporter constructs. After serum starvation overnight and 30min insulin stimulation, mRNA levels of reporter constructs were measured by qPCR and normalized to untreated (no RNAi/insulin). Data are represented by Means ± SEM from three independent experiments. (see 3B). <b>C</b>. Changes of the reporter mRNA levels by Not1 depletion in the absence of insulin stimulation. The experiment was performed as in B except the absence of insulin addition. <b>D</b>. Combined effect of insulin stimulation and Not1 depletion on the reporter mRNA levels. The mRNA levels of the indicated constructs were normalized to untreated (no RNAi/insulin).</p

Subunits of <i>Drosophila</i> Ccr4-Not complex.

<p>* See references [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113902#pone.0113902.ref014" target="_blank">14</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113902#pone.0113902.ref021" target="_blank">21</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113902#pone.0113902.ref022" target="_blank">22</a>]</p><p>Subunits of <i>Drosophila</i> Ccr4-Not complex.</p

Not1 depletion changes the mRNA level of 4E-BP.

<p>Kc cells were treated with Not1 or mock (EGFP) RNAi and mRNA levels of the indicated genes were measured by qPCR and normalized to mock RNAi. Data are represented as Means ± SEM from three independent experiments. <b>A</b>. Increased 4E-BP mRNA levels after Not1 knockdown and insulin stimulation. Kc cells were insulin-stimulated 30 min before lysis. <b>B</b>. Increased 4E-BP mRNA levels after Not1 knockdown without concomitant stimulation. <b>C</b>. Combined effect of insulin stimulation and Not1 RNAi on 4E-BP mRNA levels.</p

Not1 reduction increases 4E-BP mRNA through the 3’UTR of 4E-BP gene.

<p><b>A</b>. Schematic illustration of the reporter constructs in which the 5’UTR or 3’UTR was exchanged with the counterpart of 4E-BP gene. <b>B</b>. Changes of the reporter mRNA levels by Not1 depletion. Cells were transfected with the reporter constructs and subjected to Not1 RNAi. After serum starvation overnight and 30 min insulin stimulation, mRNA levels of reporter constructs were measured by qPCR and normalized to untreated (no RNAi/insulin). Data are represented by Means ± SEM from three independent experiments.</p

Depletion of Ccr4-Not complex components in Kc cells increases phosphorylation of 4E-BP and cell size upon insulin stimulation.

<p><b>A</b>. Increase in insulin-induced 4E-BP phosphorylation by RNAi knockdown of Ccr4-Not complex components. Kc cells were subjected to RNAi treatments against the indicated genes and stimulated with insulin 30 min before cell lysis. The lysates were analyzed by Western blotting using antibodies against anti-phospho 4E-BP and tubulin. <b>B</b>. Increased cell size caused by RNAi knockdown of Ccr4-Not complex components. Kc cells were treated as in A. The cell size (area) was measured from three independent experiments and normalized to control (EGFP RNAi). Data are presented as Means ± SEM.</p

Regulation of 4E-BP translation by Ccr4-Not complex is not detected.

<p><b>A</b>. Schematic of reporter constructs that were used for protein level assessment. <b>B</b>. Effect of Not1 reduction on the protein levels of the reporter constructs. Cells were under Not1 RNAi and transfection with the indicated constructs. After serum starvation overnight and insulin stimulation for 30 min, cells were lysed and subjected to Western blotting analysis using antibodies against GFP and tubulin. The GFP protein levels (normalized to tubulin) were densitometrically quantified (Image J) from two independent experiments and normalized to untreated (no RNAi/insulin). Means ± SEM are shown. A representative blot is shown. <b>C</b>. Relative mRNA and protein levels of the reporter constructs normalized to pAAA.</p