Hans-Joerg Hippe

Activation of Heterotrimeric G Proteins by a High Energy Phosphate Transfer via Nucleoside Diphosphate Kinase (NDPK) B and Gβ Subunits SPECIFIC ACTIVATION OF Gsα BY AN NDPK B·Gβγ COMPLEX IN H10 CELLS

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro. To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B were stably expressed in H10 cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of Gsα and Giα proteins. However, co-expression of Gsα led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited a decreased cAMP formation in response to Gsα. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with Gβγ dimers. The overexpression of NDPK B, but not its inactive mutant or NDPK A, increased the phosphorylation of Gβ subunits. In summary, our data demonstrate a specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B·Gβγ complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins.

The interaction of nucleoside diphosphate kinase B with Gβγ dimers controls heterotrimeric G protein function

Heterotrimeric G proteins in physiological and pathological processes have been extensively studied so far. However, little is known about mechanisms regulating the cellular content and compartmentalization of G proteins. Here, we show that the association of nucleoside diphosphate kinase B (NDPK B) with the G protein βγ dimer (Gβγ) is required for G protein function in vivo. In zebrafish embryos, morpholino-mediated knockdown of zebrafish NDPK B, but not NDPK A, results in a severe decrease in cardiac contractility. The depletion of NDPK B is associated with a drastic reduction in Gβ1γ2 dimer expression. Moreover, the protein levels of the adenylyl cyclase (AC)-regulating Gαs and Gαi subunits as well as the caveolae scaffold proteins caveolin-1 and -3 are strongly reduced. In addition, the knockdown of the zebrafish Gβ1 orthologs, Gβ1 and Gβ1like, causes a cardiac phenotype very similar to that of NDPK B morphants. The loss of Gβ1/Gβ1like is associated with a down-regulation in caveolins, AC-regulating Gα-subunits, and most important, NDPK B. A comparison of embryonic fibroblasts from wild-type and NDPK A/B knockout mice demonstrate a similar reduction of G protein, caveolin-1 and basal cAMP content in mammalian cells that can be rescued by re-expression of human NDPK B. Thus, our results suggest a role for the interaction of NDPK B with Gβγ dimers and caveolins in regulating membranous G protein content and maintaining normal G protein function in vivo.

Levels of circulating miR-133a are elevated in sepsis and predict mortality in critically ill patients.

Objective:Serum levels of microRNA have been proposed as biomarkers in various inflammatory diseases. However, up to now, their clinical relevance in critical illness and sepsis is unclear. Design:Single-center clinical study. Setting:Fourteen-bed medical ICU of the University Hospital Aachen, university laboratory research unit. Subjects and Patients:Experimental sepsis model in C57Bl/6 mice; 223 critically ill patients in comparison with 76 healthy volunteers. Interventions:We used the model of cecal pole ligation and puncture for induction of polymicrobial sepsis in mice and measured alterations in serum levels of six different microRNAs with a known function in inflammatory diseases upon induction of septic disease. These results from mice were translated into a large and well-characterized cohort of critically ill patients admitted to the medical ICU. Measurements and Main Results:Serum miR-133a was then measured in 223 critically ill patients (138 with sepsis and 85 without sepsis) and 76 controls and associated with disease severity, organ failure, and prognosis. Significant alterations of miR-133a, miR-150, miR-155, and miR-193b* were found in mice after cecal pole ligation and puncture–induced sepsis. Among all regulated microRNAs, miR-133a displayed the most prominent and concordant up-regulation in sepsis, and this microRNA was therefore chosen for further investigation in the human. Here, significantly elevated miR-133a levels were found in critically ill patients at ICU admission, when compared with healthy controls, especially in patients with sepsis. Correlation analyses revealed significant correlations of miR-133a with disease severity, classical markers of inflammation and bacterial infection, and organ failure. Strikingly, high miR-133a levels were predictive for an unfavorable prognosis and represented a strong independent predictor for both ICU and long-term mortality in critically ill patients. Conclusions:miR-133a serum levels were significantly elevated in critical illness and sepsis. High miR-133a levels were associated with the severity of disease and predicted an unfavorable outcome of critically ill patients.

Regulation of Cardiac cAMP Synthesis and Contractility by Nucleoside Diphosphate Kinase B/G Protein βγ Dimer Complexes

Heterotrimeric G proteins are pivotal regulators of myocardial contractility. In addition to the receptor-induced GDP/GTP exchange, G protein &agr; subunits can be activated by a phosphate transfer via a plasma membrane-associated complex of nucleoside diphosphate kinase B (NDPK B) and G protein &bgr;&ggr;-dimers (G&bgr;&ggr;). To investigate the physiological role of this phosphate transfer in cardiomyocytes, we generated a G&bgr;1&ggr;2-dimer carrying a single amino acid exchange at the intermediately phosphorylated His-266 in the &bgr;1 subunit (G&bgr;1H266L&ggr;2). Recombinantly expressed G&bgr;1H266L&ggr;2 were integrated into heterotrimeric G proteins in rat cardiomyocytes but were deficient in intermediate G&bgr; phosphorylation. Compared with wild-type G&bgr;1&ggr;2 (G&bgr;1WT&ggr;2), overexpression of G&bgr;1H266L&ggr;2 suppressed basal cAMP formation up to 55%. A similar decrease in basal cAMP production occurred when the formation of NDPK B/G&bgr;&ggr; complexes was attenuated by siRNA-mediated NDPK B knockdown. In adult rat cardiomyocytes expressing G&bgr;1H266L&ggr;2, the basal contractility was suppressed by ≈50% which correlated to similarly reduced basal cAMP levels and reduced Ser16-phosphorylation of phospholamban. In the presence of the &bgr;-adrenoceptor agonist isoproterenol, the total cAMP formation and contractility were significantly lower in G&bgr;1H266L&ggr;2 than in G&bgr;1WT&ggr;2 expressing cardiomyocytes. However, the relative isoproterenol-induced increased was not affected by G&bgr;1H266L&ggr;2. We conclude that the receptor-independent activation of G proteins via NDPK B/G&bgr;&ggr; complexes requires the intermediate phosphorylation of G protein &bgr; subunits at His-266. Our results highlight the histidine kinase activity of NDPK B for G&bgr; and demonstrate its contribution to the receptor-independent regulation of cAMP synthesis and contractility in intact cardiomyocytes.

Through scaffolding and catalytic actions nucleoside diphosphate kinase B differentially regulates basal and β-adrenoceptor-stimulated cAMP synthesis

β-adrenoceptors (βAR) play a central role in the regulation of cAMP synthesis and cardiac contractility. Nucleoside diphosphate kinase B (NDPK B) regulates cAMP signalling by complex formation with Gβγ dimers thereby activating and stabilizing heterotrimeric G(s) proteins, key transducer of βAR signals into the cell. Here, we explored the requirement of NDPK B for basal and βAR-stimulated cAMP synthesis and analysed the underlying mechanisms by comparing wild-type NDPK B (WT) and its catalytically inactive H118N mutant. Stable overexpression of both WT- and H118N-NDPK B in cardiomyocyte derived H10 cells increased the plasma membrane content of G(s) and caveolin-1 and thus enhanced the isoproterenol (ISO)-stimulated cAMP-synthesis by about 2-fold. Conversely, the loss of NDPK B in embryonic fibroblasts from NDPK A/B-depleted mice was associated with a severe reduction in membranous G(s) protein and carveolin-1 content causing a marked decrease in basal and ISO-induced cAMP formation. Re-expression of NDPK B, but not of NDPK A, was able to rescue this phenotype. Both, re-expression of WT- and H118N-NDPK B induced the re-appearance of G(s) and caveolin-1 at the plasma membrane to a similar extent. Accordingly, WT- and H118N-NDPK B similarly enhanced ISO-induced cAMP formation. In contrast, the catalytically inactive H118N-NDPK B was less potent and less effective in rescuing basal cAMP production. Identical results were obtained in neonatal rat cardiac myocytes after siRNA-induced knockdown and adenoviral re-expression of NDPK B. Our data reveal that NDPK B regulates G(s) function by two different mechanisms. The complex formation of NDPK B with G(s) is required for the stabilization of the G protein content at the plasma membrane. In addition, the NDPK B-dependent phosphotransfer reaction, which requires the catalytic activity, specifically allows a receptor-independent, basal G(s) activation.

Elevated serum levels of bone sialoprotein during ICU treatment predict long-term mortality in critically ill patients

Bone sialoprotein (BSP), a member of the SIBLINGs (for Small Integrin-Binding LIgand, N-linked Glycoproteins) family, has recently be associated to inflammatory and infectious diseases. We therefore measured BSP concentrations in 136 patients at admission to the intensive care unit (ICU) and 3 days of ICU. BSP levels were compared to 36 healthy blood donors and correlated to clinical data. In these analysis, BSP serum levels were strongly elevated at the time point of admission to the ICU when compared to healthy controls. Moreover BSP concentrations were significantly elevated after 3 days of treatment on the intensive care unit. A further increase in BSP levels was detected in patients with higher APACHE-II-scores and in patients with septic disease. While in most patients, BSP levels decreased during the first three days of treatment on a medical ICU, patients with persistently elevated BSP levels displayed an unfavorable outcome. In these patients, persistently elevated BSP concentrations were a superior predictor of mortality than established indicators of patient´ prognosis such as the SAPS2 or the APACHE-II score. In summary, our data argue for a novel utility for BSP as a biomarker in patients treated on a medical ICU.

Atypical symptoms of a fatal myocardial infarction in an elderly patient

An 83-year-old woman was admitted to our emergency department because of altered mental state and suspected exsiccosis. Family members reported that 3 days earlier, the mental state of the woman has still been unremarkable. On admission, her blood pressure was 155/92 mmHg, pulse 95 b.p.m., temperature 36.1°C, respiration rate 16 breaths/min and blood sugar 195 mg/deciliter. She was awake and responsive, oriented to people, but disoriented to time and place. She stated that she had no complaints, especially no angina pectoris or dyspnea. According to her medical history, she had a middle cerebral artery infarction 6 years earlier and arterial hypertension. Physical examination showed no functional neurological deficit and showed no evidence of exsiccosis. Auscultation of the heart found undertone heart sounds, a silent systolic murmur and discrete crackling sounds on the lungs. Of note, no peripheral edema or cyanosis were noticed on the initial examination. In our emergency department, every patient receives a resting electrocardiogram directly on admission, irrespective of the initial complaint leading to the admission. In the present patient, the electrocardiogram showed an anterior wall ST-segment elevation myocardial infarction (Fig. 1a). Troponin T was 442 pg/mL (normal <14 pg/mL), creatine kinase was 1454 U/L (normal <140 U/L), lactate dehydrogenase was 804 U/L (normal <255 U/L). In line with international guidelines on the management of ST elevation myocardial infarction, we immediately carried out a coronary angiography. This examination showed a thrombotic occlusion of the ramus interventricularis

Affixin (β-parvin) promotes cardioprotective signaling via STAT3 activation

The focal adhesion protein affixin (β-parvin) is highly expressed in the heart and is associated with the sarcomeric z-disc as well as the cell membrane. While affixin is known to be involved in cell adhesion and migration, its functional role in cardiomyocytes remains unclear. To gain insight into the function of affixin, we performed a yeast-two-hybrid-screen employing affixin as a bait. The signal transducer and activator of transcription 3 (STAT3) was detected as a binding partner of affixin. Overexpression of affixin in neonatal rat cardiomyocytes resulted in markedly enhanced STAT3 DNA binding activity and upregulation of STAT3-dependent genes. Moreover, upregulation of affixin led to cardiomyocyte hypertrophy with an increase in cell size and enhanced protein synthesis. Consistent with STAT3 activation, overexpression of affixin also protected cardiomyocytes from doxorubicin-induced apoptosis. Finally, HUVECs that were cultivated in medium from affixin-overexpressing cardiomyocytes responded with an increase in tubuli formation, in line with a proangiogenic effect of affixin. In conclusion, we demonstrate that affixin activates STAT3 in cardiomyocytes and promotes characteristic STAT3-related effects such as hypertrophy, protection against apoptosis, and angiogenesis. This novel pathway might therefore represent a target for cardioprotective strategies.