Lipopolysaccharide-induced sepsis impairs M2R-GIRK signaling in the mouse sinoatrial node.

Sepsis has emerged as a global health burden associated with multiple organ dysfunction and 20% mortality rate in patients. Numerous clinical studies over the past two decades have correlated the disease severity and mortality in septic patients with impaired heart rate variability (HRV), as a consequence of impaired chronotropic response of sinoatrial node (SAN) pacemaker activity to vagal/parasympathetic stimulation. However, the molecular mechanism(s) downstream to parasympathetic inputs have not been investigated yet in sepsis, particularly in the SAN. Based on electrocardiography, fluorescence Ca2+ imaging, electrophysiology, and protein assays from organ to subcellular level, we report that impaired muscarinic receptor subtype 2-G protein-activated inwardly-rectifying potassium channel (M2R-GIRK) signaling in a lipopolysaccharide-induced proxy septic mouse model plays a critical role in SAN pacemaking and HRV. The parasympathetic responses to a muscarinic agonist, namely IKACh activation in SAN cells, reduction in Ca2+ mobilization of SAN tissues, lowering of heart rate and increase in HRV, were profoundly attenuated upon lipopolysaccharide-induced sepsis. These functional alterations manifested as a direct consequence of reduced expression of key ion-channel components (GIRK1, GIRK4, and M2R) in the mouse SAN tissues and cells, which was further evident in the human right atrial appendages of septic patients and likely not mediated by the common proinflammatory cytokines elevated in sepsis.

Polysulfides derived from the hydrogen sulfide and persulfide donor P* inhibit IL-1ß-mediated inducible nitric oxide synthase signaling in ATDC5 cells: are CCAAT/enhancer-binding proteins ß and δ involved in the anti-inflammatory effects of hydrogen sulfide and polysulfides?

Hydrogen sulfide (H2S) emerged as an essential signaling molecule exerting beneficial effects in various cardiovascular, neurodegenerative, or musculoskeletal diseases with an inflammatory component, such as osteoarthritis. These protective effects were initially attributed to protein S-sulfhydration, a posttranslational modification of reactive cysteine residues. However, recent studies suggest that polysulfides and not H2S are responsible for S-sulfhydration. To distinguish between H2S and polysulfide-mediated effects in this study, we used the slow-releasing H2S and persulfide donor P*, which can be decomposed into polysulfides. The effects of P* on IL-1ß-induced inducible nitric oxide synthase (iNOS), a pro-inflammatory mediator in osteoarthritis, were determined by nitrite measurement, qPCR, and Western blotting in the murine chondrocyte-like cell line ATDC5. Decomposed P* significantly reduced IL-1ß-induced iNOS signaling via polysulfides, independently of H2S. In line with this, the fast-releasing H2S donor NaHS was ineffective. In RAW 264.7 macrophages, similar results were obtained. P*-derived polysulfides further diminished IL-1ß-induced CCAAT/enhancer-binding protein (C/EBP) ß and δ expression in ATDC5 cells, which might play a critical role in P*-mediated iNOS decline. In conclusion, our data support the view that polysulfides are essential signaling molecules as well as potential mediators of H2S signaling. Moreover, we propose that C/EBPß/δ might be a novel target involved in H2S and polysulfide-mediated anti-inflammatory signaling.

Influence of Embedding Fiber Optical Sensors in CFRP Film Adhesive Joints on Bond Strength.

The increasing utilization of carbon fiber reinforced plastic (CFRP) in the aeronautical industry calls for a structural health monitoring (SHM) system for adhesively bonded CFRP joints. Optical glass fiber with inscribed fiber Bragg gratings (FBGs) is a promising technology for a SHM system. This paper investigates the intrusive effect of embedding optical glass fibers carrying FBGs on adhesive bond strength and adhesive layer thickness and quality. Embedding the optical glass fibers directly in the adhesive bond has the advantage of directly monitoring the targeted structure but poses the risk of significantly reducing the bond strength. Optical glass fibers with different cladding diameters (50, 80, 125 µm) and coating types (polyimide, with a thickness of 3-8 µm, and acrylate, with a thickness of ~35 µm) are embedded in structural and repair film adhesives here. Without embedded optical glass fibers, the film adhesives have an adhesive layer thickness of ~90 µm (structural) and ~100 µm (repair) after curing. The intrusive effect of the fiber embedding on the adhesive bond strength is investigated here with quasi static and fatigue single lap joint (SLJ) tensile shear tests. Also, the influence of hydrothermal aging procedures on the quasi static tensile shear strength is investigated. It is found that optical glass fibers with a total diameter (glass fiber cladding + coating) of ~145 µm significantly reduce the quasi static tensile shear strength and increase the adhesive layer thickness and number of air inclusions (or pores) in the structural film adhesive joints. In the repair adhesive joints, no significant reduction of quasi static tensile shear strength is caused by the embedding of any of the tested fiber types and diameters. However, an increase in the adhesive layer thickness is detected. In both adhesive films, no effect on the quasi-static tensile shear strength is detected when embedding optical glass fibers with total diameters <100 µm. The applied aging regime only affects the repair film adhesive joints, and the structural film adhesive joints show no significant reduction. A polyimide-coated 80 µm optical glass fiber is selected for fatigue SLJ tensile shear tests in combination with the more sensitive structural film adhesive. No significant differences between the S-N curves and tensile shear fatigue strength of the reference samples without embedded optical fibers and the samples carrying the polyimide-coated 80 µm optical glass fibers are detected. Thus, it is concluded that the influences of embedding optical glass fibers with total diameters <100 µm on the fatigue limit of the tested film adhesive joints is negligible.

Adenosine kinase attenuates cardiomyocyte microtubule stabilization and protects against pressure overload-induced hypertrophy and LV dysfunction.

Adenosine exerts numerous protective actions in the heart, including attenuation of cardiac hypertrophy. Adenosine kinase (ADK) converts adenosine to adenosine monophosphate (AMP) and is the major route of myocardial adenosine metabolism, however, the impact of ADK activity on cardiac structure and function is unknown. To examine the role of ADK in cardiac homeostasis and adaptation to stress, conditional cardiomyocyte specific ADK knockout mice (cADK-/-) were produced using the MerCreMer-lox-P system. Within 4 weeks of ADK disruption, cADK-/- mice developed spontaneous hypertrophy and increased ß-Myosin Heavy Chain expression without observable LV dysfunction. In response to 6 weeks moderate left ventricular pressure overload (transverse aortic constriction;TAC), wild type mice (WT) exhibited ~60% increase in ventricular ADK expression and developed LV hypertrophy with preserved LV function. In contrast, cADK-/- mice exhibited significantly greater LV hypertrophy and cardiac stress marker expression (atrial natrurietic peptide and ß-Myosin Heavy Chain), LV dilation, reduced LV ejection fraction and increased pulmonary congestion. ADK disruption did not decrease protein methylation, inhibit AMPK, or worsen fibrosis, but was associated with persistently elevated mTORC1 and p44/42 ERK MAP kinase signaling and a striking increase in microtubule (MT) stabilization/detyrosination. In neonatal cardiomyocytes exposed to hypertrophic stress, 2-chloroadenosine (CADO) or adenosine treatment suppressed MT detyrosination, which was reversed by ADK inhibition with iodotubercidin or ABT-702. Conversely, adenoviral over-expression of ADK augmented CADO destabilization of MTs and potentiated CADO attenuation of cardiomyocyte hypertrophy. Together, these findings indicate a novel adenosine receptor-independent role for ADK-mediated adenosine metabolism in cardiomyocyte microtubule dynamics and protection against maladaptive hypertrophy.

Site and mechanism of uncoupling of nitric-oxide synthase: Uncoupling by monomerization and other misconceptions.

Nitric oxide synthase (NOS) catalyzes the transformation of l-arginine, molecular oxygen (O2), and NADPH-derived electrons to nitric oxide (NO) and l-citrulline. Under some conditions, however, NOS catalyzes the reduction of O2 to superoxide (O2-) instead, a phenomenon that is generally referred to as uncoupling. In principle, both the heme in the oxygenase domain and the flavins in the reductase domain could catalyze O2- formation. In the former case the oxyferrous (Fe(II)O2) complex that is formed as an intermediate during catalysis would dissociate to heme and O2-; in the latter case the reduced flavins would reduce O2 to O2-. The NOS cofactor tetrahydrobiopterin (BH4) is indispensable for coupled catalysis. In the case of uncoupling at the heme this is explained by the essential role of BH4 as an electron donor to the oxyferrous complex; in the case of uncoupling at the flavins it is assumed that the absence of BH4 results in NOS monomerization, with the monomers incapable to sustain NO synthesis but still able to support uncoupled catalysis. In spite of little supporting evidence, uncoupling at the reductase after NOS monomerization appears to be the predominant hypothesis at present. To set the record straight we extended prior studies by determining under which conditions uncoupling of the neuronal and endothelial isoforms (nNOS and eNOS) occurred and if a correlation exists between uncoupling and the monomer/dimer equilibrium. We determined the rates of coupled/uncoupled catalysis by measuring NADPH oxidation spectrophotometrically at 340 nm and citrulline synthesis as the formation of [3H]-citrulline from [3H]-Arg. The monomer/dimer equilibrium was determined by FPLC and, for comparison, by low-temperature polyacrylamide gel electrophoresis. Uncoupling occurred in the absence of Arg and/or BH4, but not in the absence of Ca2+ or calmodulin (CaM). Since omission of Ca2+/CaM will completely block heme reduction while still allowing substantial FMN reduction, this argues against uncoupling by the reductase domain. In the presence of heme-directed NOS inhibitors uncoupling occurred to the extent that these compound allowed heme reduction, again arguing in favor of uncoupling at the heme. The monomer/dimer equilibrium showed no correlation with uncoupling. We conclude that uncoupling by BH4 deficiency takes place exclusively at the heme, with virtually no contribution from the flavins and no role for NOS monomerization.

Irreversible Activation and Stabilization of Soluble Guanylate Cyclase by the Protoporphyrin IX Mimetic Cinaciguat.

Belonging to the class of so-called soluble guanylate cyclase (sGC) activators, cinaciguat and BAY 60-2770 are interesting therapeutic tools for the treatment of various cardiovascular pathologies. The drugs are supposed to preferentially stimulate oxidized or heme-depleted, but not native sGC. Since this concept has been challenged by studies demonstrating complete relaxation of nondiseased vessels, this study was designed to reinvestigate the mode of action in greater detail. To this purpose, the effect of cinaciguat was studied on vessel tone of porcine coronary arteries and rat thoracic aortas. Organ bath studies showed that the compound caused time- and concentration-dependent relaxation of precontracted vessels with a maximal effect observed at 90 minutes. The dilatory response was not affected by extensive washout of the drug. Cinaciguat-induced vasodilation was associated with a time- and concentration-dependent increase of cGMP levels. Experiments with purified sGC in the presence of Tween 20 showed that cinaciguat activates the heme-free enzyme in a concentration-dependent manner with an EC50 value of ∼0.2 µM and maximal cGMP formation at 10 µM. By contrast, the effect of cinaciguat on 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one-oxidized (ferric) sGC was moderate, reaching ∼10%-15% of maximal activity. Dilution experiments of cinaciguat/Tween 20-preincubated sGC revealed the irreversible character of the drug. Assuming a sensitive balance between heme-free, ferric, and nitric oxide-sensitive ferrous sGC in cells and tissues, we propose that cinaciguat by virtue of its irreversible mode of action is capable of shifting this equilibrium toward the heme-free apo-sGC species.

Sustained Formation of Nitroglycerin-Derived Nitric Oxide by Aldehyde Dehydrogenase-2 in Vascular Smooth Muscle without Added Reductants: Implications for the Development of Nitrate Tolerance.

According to current views, oxidation of aldehyde dehydrogenase-2 (ALDH2) during glyceryltrinitrate (GTN) biotransformation is essentially involved in vascular nitrate tolerance and explains the dependence of this reaction on added thiols. Using a novel fluorescent intracellular nitric oxide (NO) probe expressed in vascular smooth muscle cells (VSMCs), we observed ALDH2-catalyzed formation of NO from GTN in the presence of exogenously added dithiothreitol (DTT), whereas only a short burst of NO, corresponding to a single turnover of ALDH2, occurred in the absence of DTT. This short burst of NO associated with oxidation of the reactive C302 residue in the active site was followed by formation of low-nanomolar NO, even without added DTT, indicating slow recovery of ALDH2 activity by an endogenous reductant. In addition to the thiol-reversible oxidation of ALDH2, thiol-refractive inactivation was observed, particularly under high-turnover conditions. Organ bath experiments with rat aortas showed that relaxation by GTN lasted longer than that caused by the NO donor diethylamine/NONOate, in line with the long-lasting nanomolar NO generation from GTN observed in VSMCs. Our results suggest that an endogenous reductant with low efficiency allows sustained generation of GTN-derived NO in the low-nanomolar range that is sufficient for vascular relaxation. On a longer time scale, mechanism-based, thiol-refractive irreversible inactivation of ALDH2, and possibly depletion of the endogenous reductant, will render blood vessels tolerant to GTN. Accordingly, full reactivation of oxidized ALDH2 may not occur in vivo and may not be necessary to explain GTN-induced vasodilation.

Formation of Nitric Oxide by Aldehyde Dehydrogenase-2 Is Necessary and Sufficient for Vascular Bioactivation of Nitroglycerin.

Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 µm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 µm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but lacks clearance-based GTN denitration activity. NO formation was compared with GTN-induced activation of sGC. The addition of 1 µm GTN to VSMC expressing either wild-type or C301S/C303S ALDH2 resulted in pronounced intracellular NO elevation, with maximal concentrations of 7 and 17 nm, respectively. Formation of GTN-derived NO correlated well with activation of purified sGC in VSMC lysates and cGMP accumulation in intact porcine aortic endothelial cells infected with wild-type or mutant ALDH2. Formation of NO and cGMP accumulation were inhibited by ALDH inhibitors chloral hydrate and daidzin. The present study demonstrates that ALDH2-catalyzed NO formation is necessary and sufficient for GTN bioactivation in VSMC.

Real-time visualization of distinct nitric oxide generation of nitric oxide synthase isoforms in single cells.

The members of the nitric oxide synthase (NOS) family, eNOS, nNOS and iNOS, are well-characterized enzymes. However, due to the lack of suitable direct NO sensors, little is known about the kinetic properties of cellular NO generation by the different nitric oxide synthase isoenzymes. Very recently, we developed a novel class of fluorescent protein-based NO-probes, the geNOps, which allow real-time measurement of cellular NO generation and fluctuation. By applying these genetic NO biosensors to nNOS-, eNOS- and iNOS-expressing HEK293 cells we were able to characterize the respective NO dynamics in single cells that exhibited identical Ca2+ signaling as comparable activator of nNOS and eNOS. Our data demonstrate that upon Ca2+ mobilization nNOS-derived NO signals occur instantly and strictly follow the Ca2+ elevation while NO release by eNOS occurs gradually and sustained. To detect high NO levels in cells expressing iNOS, a new ratiometric probe based on two fluorescent proteins was developed. This novel geNOp variant allows the measurement of the high NO levels in cells expressing iNOS. Moreover, we used this probe to study the L-arginine-dependency of NO generation by iNOS on the level of single cells. Our experiments highlight that the geNOps technology is suitable to detect obvious differences in the kinetics, amplitude and substrate-dependence of cellular NO signals-derived from all three nitric oxide synthase isoforms.

Using systems biology to evaluate targets and mechanism of action of drugs for diabetes comorbidities.

Medications approved for diabetes-associated renal and cardiovascular morbidities and candidate drugs currently in development are subject to substantial variability in drug response. Heterogeneity on a molecular phenotype level is not apparent at clinical presentation, which means that inter-individual differences in drug effect at the molecular level are masked. These findings identify the need for optimising patient phenotyping via use of molecular biomarkers for a personalised therapy approach. Molecular diversity may, on the one hand, result from the effect of genetic polymorphisms on drug transport, metabolism and effective target modulation. Equally relevant, differences may be due to molecular pathologies. The presence of distinct molecular phenotypes is suggested by classifiers aimed at modelling progressive disease. Such functions for prognosis incorporate a complex set of clinical variables or a multitude of molecular markers reflecting a diverse set of molecular disease mechanisms. This information on disease pathology and the mechanism of action of the drug needs to be systematically integrated with data on molecular biomarkers to develop an experimental tool for personalising medicine. The large amount of molecular data available for characterising diabetes-associated morbidities allows for elucidation of molecular process model representations of disease pathologies. Selecting biomarker candidates on such grounds and, in turn identifying their association with progressive disease allows for the identification of molecular processes associated with disease progression. The molecular effect of a drug can also be modelled at a molecular process level, and the integration of disease pathology and drug effect molecular models reveals candidate biomarkers for assessing drug response. Such tools serve as enrichment strategies aimed at adding precision to drug development and use.