Ulrich Flögel

Nitrite Reductase Function of Deoxymyoglobin Oxygen Sensor and Regulator of Cardiac Energetics and Function

Although the primary function of myoglobin (Mb) has been considered to be cellular oxygen storage and supply, recent studies have suggested to classify Mb as a multifunctional allosteric enzyme. In the heart, Mb acts as a potent scavenger of nitric oxide (NO) and contributes to the attenuation of oxidative damage. Here we report that a dynamic cycle exists in which a decrease in tissue oxygen tension drives the conversion of Mb from being an NO scavenger in normoxia to an NO producer in hypoxia. The NO generated by reaction of deoxygenated Mb with nitrite is functionally relevant and leads to a downregulation of cardiac energy status, which was not observed in mice lacking Mb. As a consequence, myocardial oxygen consumption is reduced and cardiac contractility is dampened in wild-type mice. We propose that this pathway represents a novel homeostatic mechanism by which a mismatch between oxygen supply and demand in muscle is translated into the fractional increase of deoxygenated Mb exhibiting enhanced nitrite reductase activity. Thus, Mb may act as an oxygen sensor which through NO can adjust muscle energetics to limited oxygen supply.

In Vivo Monitoring of Inflammation After Cardiac and Cerebral Ischemia by Fluorine Magnetic Resonance Imaging

Background— In this study, we developed and validated a new approach for in vivo visualization of inflammatory processes by magnetic resonance imaging using biochemically inert nanoemulsions of perfluorocarbons (PFCs). Methods and Results— Local inflammation was provoked in 2 separate murine models of acute cardiac and cerebral ischemia, followed by intravenous injection of PFCs. Simultaneous acquisition of morphologically matching proton (1H) and fluorine (19F) images enabled an exact anatomic localization of PFCs after application. Repetitive 1H/19F magnetic resonance imaging at 9.4 T revealed a time-dependent infiltration of injected PFCs into the border zone of infarcted areas in both injury models, and histology demonstrated a colocalization of PFCs with cells of the monocyte/macrophage system. We regularly found the accumulation of PFCs in lymph nodes. Using rhodamine-labeled PFCs, we identified circulating monocytes/macrophages as the main cell fraction taking up injected nanoparticles. Conclusions— PFCs can serve as a “positive” contrast agent for the detection of inflammation by magnetic resonance imaging, permitting a spatial resolution close to the anatomic 1H image and an excellent degree of specificity resulting from the lack of any 19F background. Because PFCs are nontoxic, this approach may have a broad application in the imaging and diagnosis of numerous inflammatory disease states.

Targeted Disruption of cd73/Ecto-5′-Nucleotidase Alters Thromboregulation and Augments Vascular Inflammatory Response

To investigate the role of adenosine formed extracellularly in vascular homeostasis, mice with a targeted deletion of the cd73/ecto-5′-nucleotidase were generated. Southern blot, RT-PCR, and Western blot analysis confirmed the constitutive knockout. In vivo analysis of hemodynamic parameters revealed no significant differences in systolic blood pressure, ejection fraction, or cardiac output between strains. However, basal coronary flow measured in the isolated perfused heart was significantly lower (−14%; P<0.05) in the mutant. Immunohistochemistry revealed strong CD73 expression on the endothelium of conduit vessels in wild-type (WT) mice. Time to carotid artery occlusion after ferric chloride (FeCl3) was significantly reduced by 20% in cd73−/− mice (P<0.05). Bleeding time after tail tip resection tended to be shorter in cd73−/− mice (−35%). In vivo platelet cAMP levels were 0.96±0.46 in WT versus 0.68±0.27 pmol/106 cells in cd73−/− mice (P<0.05). Under in vitro conditions, platelet aggregation in response to ADP (0.05 to 10 &mgr;mol/L) was undistinguishable between the two strains. In the cremaster model of ischemia–reperfusion, the increase in leukocyte attachment to endothelium was significantly higher in cd73−/− compared with WT littermates (WT 98% versus cd73−/− 245%; P<0.005). The constitutive adhesion of monocytes in ex vivo–perfused carotid arteries of WT mice was negligible but significantly increased in arteries of cd73−/− mice (P<0.05). Thus, our data provide the first evidence that adenosine, extracellularly formed by CD73, can modulate coronary vascular tone, inhibit platelet activation, and play an important role in leukocyte adhesion to the vascular endothelium in vivo.

Restricted diffusion and exchange of intracellular water: theoretical modelling and diffusion time dependence of 1H NMR measurements on perfused glial cells

Intracellular diffusion properties of water in F98 glioma cells immobilized in basement membrane gel threads, are investigated with a pulsed‐field‐gradient spin‐echo NMR technique at diffusion times from 6 to 2000 ms and at different temperatures. In extended model calculations the concept of ‘restricted intracellular diffusion at permeable boundaries’ is described by a combined Tanner–Kärger formula. Signal components in a series of ct experiments (constant diffusion time) are separated due to different diffusion properties (Gaussian and restricted diffusion), and physiological as well as morphological cell parameters are extracted from the experimental data. The intracellular apparent diffusion coefficients strongly depend on the diffusion time and are up to two orders of magnitude smaller than the self diffusion constant of water. Propagation lengths are found to be in the range of 4–7 μm. Hereby intracellular signals of compartments with a characteristic diameter could be selected by an appropriate gradient strength. With cg experiments (constant gradient) a mean intracellular residence time for water is determined to be about 50 ms, and the intrinsic intracellular diffusion constant is estimated to 1 × 10−3  mm2 /s. Studying the water diffusion in glial cells provides basic understanding of the intracellular situation in brain tissue and may elucidate possible influences on the changes in the diffusion contrast during ischemic conditions.

Taurine transporter knockout depletes muscle taurine levels and results in severe skeletal muscle impairment but leaves cardiac function uncompromised

Taurine is the most abundant free amino acid in heart and skeletal muscle. In the present study, the effects of hereditary taurine deficiency on muscle function were examined in taurine transporter knockout (taut−/−) mice. These mice show an almost complete depletion of heart and skeletal muscle taurine levels. Treadmill experiments demonstrated that total exercise capacity of taut−/− mice was reduced by >80% compared with wild‐type controls. The decreased performance of taut−/− mice correlated with increased lactate levels in serum during exercise. Surprisingly, cardiac function of taut−/− mice as assessed by magnetic resonance imaging, echocardiography, and isolated heart studies showed a largely normal phenotype under both control and stimulated conditions. However, analysis of taut−/− skeletal muscle revealed electromyographic abnormalities. 1H nuclear magnetic resonance spectroscopy of tissue extracts showed that in the heart of taut−/− mice the lack of taurine was compensated by the up‐regulation of various organic solutes. In contrast, a deficit of >10 mM in total organic osmolyte concentration was found in skeletal muscle. The present study identifies taurine transport as a crucial factor for the maintenance of skeletal muscle function and total exercise capacity, while cardiac muscle apparently can compensate for the loss of taurine.

Role of myoglobin in the antioxidant defense of the heart

Although the primary function of myoglobin (Mb) has been considered to be cellular O2 storage and supply, recent studies have shown that Mb in addition can act as NO oxidase. Here we report that Mb also significantly contributes to the attenuation of oxidative stress in cardiac muscle. In support of this hypothesis, we found that in isolated perfused hearts of Mb‐deficient (myo‒/‒) mice oxidative challenge by intracoronary infused H2O2 (1‐300 µM) or superoxide formed by 2,3‐dimethoxy‐1,4‐naphtoquinone (0.1‐30 µM), respectively, depressed cardiac contractility to a greater extent than in wild‐type (WT) hearts, e.g., up to [H2O2] = 10 µM there was a significant left ventricular developed pressure (LVDP) decrease in myo‒/‒ hearts only (90.4±4.2 vs. 98.1±0.7% of control, n=6, P<0.05). Likewise in an ischemia/reperfusion protocol, myo‒/‒ hearts showed a delayed recovery of postischemic function as compared with WT controls (e.g., LVDP was 35.6±7.5 vs. 22.4±5.3 mmHg, respectively, after 10 min of reperfusion, P<0.05, n=8), which correlated well with an enhanced release of reactive oxygen species in myo‒/‒ hearts as measured by online lucigenin‐enhanced chemiluminescence [e.g. 465±87 relative light units (RLU) in myo‒/‒ vs. 287±73 RLU in WT after 2.5 min of reperfusion, P<0.05, n=8]. 31P NMR spectroscopy revealed concomitantly a more pronounced phosphocreatine overshoot during reperfusion in the knockout but only minute alterations in ATP and pHi. Our data show that lack of Mb leads to increased vulnerability of cardiac function to oxidative challenge either pharmacologically induced or endogenously generated. We propose that Mb is a key element influencing redox pathways in cardiac muscle to functionally and metabolically protect the heart from oxidative damage.

RIP3, a kinase promoting necroptotic cell death, mediates adverse remodelling after myocardial infarction.

AIMS Programmed necrosis (necroptosis) represents a newly identified mechanism of cell death combining features of both apoptosis and necrosis. Like apoptosis, necroptosis is tightly regulated by distinct signalling pathways. A key regulatory role in programmed necrosis has been attributed to interactions of the receptor-interacting protein kinases, RIP1 and RIP3. However, the specific functional role of RIP3-dependent signalling and necroptosis in the heart is unknown. The aims of this study were thus to assess the significance of necroptosis and RIP3 in the context of myocardial ischaemia. METHODS AND RESULTS Immunoblots revealed strong expression of RIP3 in murine hearts, indicating potential functional significance of this protein in the myocardium. Consistent with a role in promoting necroptosis, adenoviral overexpression of RIP3 in neonatal rat cardiomyocytes and stimulation with TNF-α induced the formation of a complex of RIP1 and RIP3. Moreover, RIP3 overexpression was sufficient to induce necroptosis of cardiomyocytes. In vivo, cardiac expression of RIP3 was up-regulated upon myocardial infarction (MI). Conversely, mice deficient for RIP3 (RIP3(-/-)) showed a significantly better ejection fraction (45 ± 3.6 vs. 32 ± 4.4%, P < 0.05) and less hypertrophy in magnetic resonance imaging studies 30 days after experimental infarction due to left anterior descending coronary artery ligation. This was accompanied by a diminished inflammatory response of infarcted hearts and decreased generation of reactive oxygen species. CONCLUSION Here, we show that RIP3-dependent necroptosis modulates post-ischaemic adverse remodelling in a mouse model of MI. This novel signalling pathway may thus be an attractive target for future therapies that aim to limit the adverse consequences of myocardial ischaemia.

Acute Inhibition of Myoglobin Impairs Contractility and Energy State of iNOS-Overexpressing Hearts

Abstract— Elevated cardiac levels of nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) have been implicated in the development of heart failure. The surprisingly benign phenotype of recently generated mice with cardiac-specific iNOS overexpression (TGiNOS) provided the rationale to investigate whether NO scavenging by oxymyoglobin (MbO2) yielding nitrate and metmyoglobin (metMb) is involved in preservation of myocardial function in TGiNOS mice. 1H nuclear magnetic resonance (NMR) spectroscopy was used to monitor changes of cardiac myoglobin (Mb) metabolism in isolated hearts of wild-type (WT) and TGiNOS mice. NO formation by iNOS resulted in a significant decrease of the MbO2 signal and a concomitantly emerging metMb signal in spectra of TGiNOS hearts only (&Dgr;MbO2: −46.3±38.4 &mgr;mol/kg, &Dgr;metMb: +41.4±17.6 &mgr;mol/kg, n=6; P <0.05) leaving contractility and energetics unaffected. Inhibition of the Mb-mediated NO degradation by carbon monoxide (20%) led to a deterioration of myocardial contractility in TGiNOS hearts (left ventricular developed pressure: 78.2±8.2% versus 96.7±4.6% of baseline, n=6; P <0.005), which was associated with a profound pertubation of cardiac energy state as assessed by 31P NMR spectroscopy (eg, phosphocreatine: 13.3±1.3 mmol/L (TGiNOS) versus 15.9±0.7 mmol/L (WT), n=6; P <0.005). These alterations could be fully antagonized by the NOS inhibitor S-ethylisothiourea. Our findings demonstrate that myoglobin serves as an important cytoplasmic buffer of iNOS-derived NO, which determines the functional consequences of iNOS overexpression.

Chronic liver disease is triggered by taurine transporter knockout in the mouse

Taurine is an abundant organic osmolyte with antioxidant and immunomodulatory properties. Its role in the pathogenesis of chronic liver disease is unknown. The liver phenotype was studied in taurine transporter knockout (taut?/?) mice. Hepatic taurine levels were ~21, 15 and 6 ?mol/g liver wet weight in adult wild‐type, heterozygous (taut+/?) and homozygous (taut?/?) mice, respectively. Immunoelectronmicroscopy revealed an almost complete depletion of taurine in Kupffer and sinusoidal endothelial cells, but not in parenchymal cells of (taut?/?) mice. Compared with wild‐type mice, (taut?/?) and (taut+/?) mice developed moderate unspecific hepatitis and liver fibrosis with increased frequency of neoplastic lesions beyond 1 year of age. Liver disease in (taut?/?) mice was characterized by hepatocyte apoptosis, activation of the CD95 system, elevated plasma TNF‐? levels, hepatic stellate cell and oval cell proliferation, and severe mitochondrial abnormalities in liver parenchymal cells. Mitochondrial dysfunction was suggested by a significantly lower respiratory control ratio in isolated mitochondria from (taut?/?) mice. Taut knockout had no effect on taurine‐conjugated bile acids in bile; however, the relative amount of cholate‐conjugates acid was decreased at the expense of 7‐keto‐cholateconjugates. In conclusion, taurine deficiency due to defective taurine transport triggers chronic liver disease, which may involve mitochondrial dysfunction.

Early Assessment of Pulmonary Inflammation by 19F MRI In Vivo

Background—Emulsified perfluorocarbons (PFCs) are preferentially phagocytized by monocytes/macrophages and are readily detected by 19F MRI. This study tests the hypothesis that 19F MRI can be used to quantitate pulmonary inflammation by tracking of infiltrating PFC-loaded monocytes. Methods and Results—Pneumonia was induced in mice by intratracheal instillation of lipopolysaccharides (LPS) followed by intravenous injection of PFCs. Whereas regular 1H MRI provided no evidence of lung injury 24 hours after LPS, the concurrent 19F images clearly show PFC accumulation in both pulmonary lobes. Imaging at 48 hours after LPS revealed signals in 1H images at the same location as the 24-hour 19F signals. Thus, progressive pneumonia was first predicted by 19F MRI early after PFC administration. Without LPS, at no time were 19F signals observed within the lung. Histology and fluorescence-activated cell sorting (FACS) combined with 19F MRI confirmed the presence of infiltrating PFC-loaded monocytes/macrophages after LPS challenge. Additional experiments with graded doses of LPS demonstrated that 19F signal intensity strongly correlated with both LPS dose and pathological markers of lung inflammation. In separate studies, dexamethasone and CGS21680 (adenosine 2A receptor agonist) were used to demonstrate the ability of 19F MRI to monitor anti-inflammatory therapies. Conclusions—PFCs serve as a contrast agent for the prognostic and quantitative assessment of pulmonary inflammation by in vivo 19F MRI, which is characterized by a high degree of specificity due to the lack of any 19F background. Because PFCs are biochemically inert, this approach may also be suitable for human applications.