The orphan receptor GPRC5B modulates inflammatory and fibrotic pathways in cardiac fibroblasts and mice hearts.

Inflammation is a major driver of cardiac remodeling. Cardiac fibroblasts play an integral role in cardiac inflammation, fibrosis and remodeling. The orphan G-protein-coupled-receptor 5B of family C (GPRC5B) has recently been shown to have pro-inflammatory effects in adipocytes via the NFκB-signaling-pathway. Here, we investigated whether GPRC5B is involved in myocardial inflammation and fibrosis. Using neonatal rat cardiac fibroblasts (NRCF) we show that the transcription and the expression of endogenous GPRC5B is induced by stimulation with TNFα and LPS as well as through cyclic mechanical stretch, while the principle pro-fibrotic factor TGFß has no effect on the GPRC5B expression. Furthermore, we demonstrate that adenoviral overexpression and siRNA-mediated knockdown of GPRC5B in NRCF significantly alters the transcription level of the pro-inflammatory and pro-fibrotic cytokines TNFα, IL-1ß, IL-6 and MCP-1, and extracellular matrix-degrading MMP-9 in vitro. Additionally, in adult GPRC5B-transgenic mice the protein expression of collagen-1A1 is decreased and the production of MMP-9 is increased, indicating remodeling of the extracellular matrix in vivo. Our data show that GPRC5B is up-regulated by inflammatory signals and mechanical stress in NRCF, while GPRC5B modulates the inflammatory response of cardiac fibroblasts and the degradation of extracellular matrix-proteins in the mice heart. Thus, our findings are the first to report a novel role of the orphan receptor GPRC5B in fibroblast-driven myocardial inflammation and cardiac remodeling.

miR-155 Predicts Long-Term Mortality in Critically Ill Patients Younger than 65 Years.

INTRODUCTION: Alterations in miR-155 serum levels have been described in inflammatory and infectious diseases. Moreover, a role for miR-155 in aging and age-related diseases was recently suggested. We therefore analyzed a potential age-dependent prognostic value of circulating miR-155 as a serum-based marker in critical illness. METHODS: Concentrations of circulating miR-155 were determined in 218 critically ill patients and 76 healthy controls. RESULTS: By using qPCR, we demonstrate that miR-155 serum levels are elevated in patients with critical illness when compared to controls. Notably, levels of circulating miR-155 were independent on the severity of disease, the disease etiology, or the presence of sepsis. In the total cohort, miR-155 was not an indicator for patient survival. Intriguingly, when patients were subdivided according to their age upon admission to the ICU into those younger than 65 years, lower levels of miR-155 turned out as a strong marker, indicating patient mortality with a similar accuracy than other markers frequently used to evaluate critically ill patients on a medical ICU. CONCLUSION: In summary, the data provided within this study suggest an age-specific role of miR-155 as a prognostic biomarker in patients younger than 65 years. Our study is the first to describe an age-dependent miRNA-based prognostic biomarker in human diseases.

Persistently elevated osteopontin serum levels predict mortality in critically ill patients.

INTRODUCTION: Inflammatory, autoimmune and metabolic disorders have been associated with alterations in osteopontin (OPN) serum levels. Furthermore, elevated serum levels of OPN were reported from a small cohort of patients with sepsis. We therefore analyzed OPN serum concentrations in a large cohort of critically ill medical patients. METHODS: A total of 159 patients (114 with sepsis, 45 without sepsis) were studied prospectively upon admission to the medical intensive care unit (ICU) as well as after 3 days of ICU treatment and compared to 50 healthy controls. Clinical data, various laboratory parameters as well as investigational inflammatory cytokine profiles were assessed. Patients were followed for approximately 1 year. RESULTS: We found significantly elevated serum levels of OPN at admission to the ICU and after 3 days of treatment in critically ill patients compared to healthy controls. OPN concentrations were related to disease severity and significantly correlated with established prognosis scores and classical as well as experimental markers of inflammation and multi-organ failure. In the total cohort, OPN levels decreased from admission to day 3 of ICU treatment. However, persistently elevated OPN levels at day 3 of ICU treatment were a strong independent predictor for an unfavorable prognosis, with similar or better diagnostic accuracy than routinely used markers of organ failure or prognostic scoring systems such as SAPS2 or APACHE II score. CONCLUSIONS: Persistently elevated OPN serum concentrations are associated with an unfavourable outcome in patients with critical illness, independent of the presence of sepsis. Besides a possible pathogenic role of OPN in critical illness, our study indicates a potential value for OPN as a prognostic biomarker in critically ill patients during the early course of ICU treatment.

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.

The interaction of nucleoside diphosphate kinase B with Gbetagamma 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 betagamma dimer (Gbetagamma) 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 Gbeta(1)gamma(2) dimer expression. Moreover, the protein levels of the adenylyl cyclase (AC)-regulating Galpha(s) and Galpha(i) subunits as well as the caveolae scaffold proteins caveolin-1 and -3 are strongly reduced. In addition, the knockdown of the zebrafish Gbeta(1) orthologs, Gbeta(1) and Gbeta(1like), causes a cardiac phenotype very similar to that of NDPK B morphants. The loss of Gbeta(1)/Gbeta(1like) is associated with a down-regulation in caveolins, AC-regulating Galpha-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 Gbetagamma dimers and caveolins in regulating membranous G protein content and maintaining normal G protein function in vivo.

Pulmonary hypertension is associated with an increased incidence of cancer diagnoses.

Pulmonary hypertension (PH) is a complex disease with increasing global incidence that eventually leads to right ventricular failure and is associated with a poor prognosis. The importance of noncardiac comorbidities in disease progression and prognosis has gained increasing recognition in recent years. In the present study, we investigated a potential association between PH and cancer in an outpatient cohort in Germany. Using the IQVIA Disease Analyzer database, we identified a total of 11,109 patients with PH and a propensity score matched cohort of equal size without PH who received medical treatment between 2005 and 2019. Logistic regression models were used to evaluate the potential association between PH and cancer. Within the 10-year observation period, the incidence of cancer was significantly higher in PH patients than non-PH patients (23.2% vs. 8.5%, log-rank p < 0.001). Importantly, this association was observed for both male (HR = 1.24, p = 0.002) and female (HR = 1.37, p < 0.001) patients, and was most pronounced in patients >80 years (HR = 1.50, p < 0.001). In terms of a specific tumor site, we found a significant association for respiratory organ cancer (HR = 1.60, p = 0.007) and skin cancer (HR = 1.48, p < 0.001). Our study provides strong evidence that PH is associated with an increased incidence of cancer. This finding should help raise awareness of this important comorbidity and could trigger specific screening programs in patients with PH.

Affixin (ß-parvin) promotes cardioprotective signaling via STAT3 activation.

The focal adhesion protein affixin (ß-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.

Regulation of cardiac cAMP synthesis and contractility by nucleoside diphosphate kinase B/G protein beta gamma dimer complexes.

Heterotrimeric G proteins are pivotal regulators of myocardial contractility. In addition to the receptor-induced GDP/GTP exchange, G protein alpha subunits can be activated by a phosphate transfer via a plasma membrane-associated complex of nucleoside diphosphate kinase B (NDPK B) and G protein betagamma-dimers (Gbetagamma). To investigate the physiological role of this phosphate transfer in cardiomyocytes, we generated a Gbeta1gamma2-dimer carrying a single amino acid exchange at the intermediately phosphorylated His-266 in the beta1 subunit (Gbeta1H266Lgamma2). Recombinantly expressed Gbeta1H266Lgamma2 were integrated into heterotrimeric G proteins in rat cardiomyocytes but were deficient in intermediate Gbeta phosphorylation. Compared with wild-type Gbeta1gamma2 (Gbeta1WTgamma2), overexpression of Gbeta1H266Lgamma2 suppressed basal cAMP formation up to 55%. A similar decrease in basal cAMP production occurred when the formation of NDPK B/Gbetagamma complexes was attenuated by siRNA-mediated NDPK B knockdown. In adult rat cardiomyocytes expressing Gbeta1H266Lgamma2, the basal contractility was suppressed by approximately 50% which correlated to similarly reduced basal cAMP levels and reduced Ser16-phosphorylation of phospholamban. In the presence of the beta-adrenoceptor agonist isoproterenol, the total cAMP formation and contractility were significantly lower in Gbeta1H266Lgamma2 than in Gbeta1WTgamma2 expressing cardiomyocytes. However, the relative isoproterenol-induced increased was not affected by Gbeta1H266Lgamma2. We conclude that the receptor-independent activation of G proteins via NDPK B/Gbetagamma complexes requires the intermediate phosphorylation of G protein beta subunits at His-266. Our results highlight the histidine kinase activity of NDPK B for Gbeta and demonstrate its contribution to the receptor-independent regulation of cAMP synthesis and contractility in intact cardiomyocytes.

Radial access protects from contrast media induced nephropathy after cardiac catheterization procedures.

OBJECTIVES: To assess, whether cardiac catheterization via radial access prevents contrast-induced nephropathy. BACKGROUND: Contrast-induced nephropathy (CIN) is a major clinical problem which accounts for more than 10% of acute kidney injury cases in hospitalized patients. Protective measures such as the infusion of isotonic saline solution or acetylcysteine have not consistently been proven to prevent acute kidney injury (AKI). However, there is growing evidence that radial access for coronary angiography and coronary intervention is associated with a lower incidence of AKI compared to femoral access. METHODS AND RESULTS: In a retrospective monocentric analysis, 2937 patients that had undergone cardiac catheterization were examined. Up to 2013, coronary intervention was performed primarily via the femoral artery in our hospital; thereafter, interventions were primarily done via the radial artery. In the cohort under study, 1141 patients had received catheterization using the radial access while 1796 were examined via the femoral artery. No significant differences were found in the two groups regarding the amount of iodinated contrast medium applied [femoral group: 180 (120-260) ml; radial group: 180 (120-250) ml; P = 0.438]. A total of 400 (13.6%) patients developed acute kidney injury (AKI) after cardiac catheterization (85.3% AKI stage 1; 12.8% AKI stage 2; 2% AKI stage 3). AKI was significantly less frequent in patients that had received radial access compared to patients with femoral access (10.1 vs. 15.9%, P < 0.001). Multivariate regression analysis showed that patient age (1.03/year; 95% CI 1.02-1.04/year; P < 0.001), the amount of contrast media applied (OR 1.003/ml; 95% CI 1.002-1.004/ml; P < 0.001), acute coronary syndrome (OR 2.01, 95% CI 1.52-2.66; P < 0.001), CKD (OR 1.62, 95% CI 1.50-1.70; P < 0.001), pre-existing heart failure (OR 1.27, 95% CI 1.00-1.42 P = 0.007), previous myocardial infarction (OR 1.34, 95% CI 1.15-1.49; P = 0.001), diabetes (OR 1.25, 95% CI 1.04-1.41; P = 0.020) and serum creatinine before the procedure (1.45/mg/dl; 95% CI 1.24-1.69/mg/dl; P < 0.001) were important risk factors for the occurrence of AKI. Our analysis points to a significant risk reduction using radial access (OR 0.65; 95% CI 0.51-0.83; P < 0.001). Interestingly, this reduction in risk was also evident in patients with CKD (OR 0.59; 95% CI 0.41-0.87; P = 0.007). The superiority of radial access was particularly obvious in the subgroup of patients with acute coronary syndrome (13.1% AKI in the radial access group vs. 23.6% AKI in the femoral access group, OR 0.52; 95% CI 0.34-0.81; P = 0.003). CONCLUSION: Our study shows that cardiac catheterization using radial access bears significantly lower risk of AKI than cardiac catheterization via femoral access. The advantage of radial access in acute coronary syndrome regarding morbidity and mortality could partly be explained by the here demonstrated reduced risk for AKI. Thus, radial access should be preferred in patients at risk for AKI.

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.

Serum levels of kisspeptin are elevated in critically ill patients.

INTRODUCTION: Members of the adipokine family such as resistin, adiponectin and omentin have recently been described as novel biomarkers with a diagnostic and prognostic role in the context of critically ill patients during intensive care unit (ICU) treatment. Kisspeptin represent another member of this family and has been shown to be closely correlated to different members of the adipokine family in manifold diseases. However, its role in critical illness and sepsis is currently unknown. MATERIALS AND METHODS: Kisspeptin serum concentrations were measured in 133 ICU patients admitted to the medical ICU. Results were compared with 36 healthy controls. RESULTS: Kisspeptin serum levels were elevated in the serum of critically ill patients at admission to the ICU, when compared to healthy controls, and remained increased after 72 hours of ICU treatment. Notably, kisspeptin levels were independent of the presence of sepsis and etiology of critical illness. In line, serum concentrations of kisspeptin were not correlated to concentrations of inflammatory cytokines or established sepsis markers. Serum kisspeptin correlated inversely with the glomerular filtration rate. In contrast to the reported role of other members of the adipokine family, serum levels of kisspeptin were neither predictive for short term survival during ICU treatment nor for patients' overall survival. Kisspeptin levels did not correlate with other adipokines measured in serum, including leptin, resistin, ghrelin, or adiponectin. CONCLUSIONS: Although circulating kisspeptin levels were strongly elevated in ICU-patients, elevated kisspeptin levels were not predictive for an impaired patients' survival.

Serum Levels of TNF Receptor Ligands Are Dysregulated in Sepsis and Predict Mortality in Critically Ill Patients.

INTRODUCTION: TNF superfamily members, including TNF-related weak inducer of apoptosis (TWEAK) and Glucocorticoid-Induced TNFR-Related Protein Ligand (GITRL) have been described as serum based biomarkers for inflammatory and immune mediated diseases. However, up to now the role of TWEAK and GITRL has not been analyzed in critical illness and sepsis. METHODS: GITRL and TWEAK serum concentrations were measured in 121 critically ill patients (84 fulfilled with septic disease), in comparison to 50 healthy controls. Results were correlated with clinical data. RESULTS: Serum levels of TWEAK and GITRL were strongly decreased in critically ill patients compared with healthy controls. Concentrations of TWEAK (but not GITRL) were further decreased in patients with sepsis and correlated with routinely used markers of inflammation and bacterial infection such as C-reactive protein, procalcitonin and Interleukin-6. Notably, we failed to detect a correlation to other TNFR ligands such as TNF or APRIL. Finally, TWEAK levels of the upper quartile of the cohort were prognostic for mortality during ICU treatment. CONCLUSION: TWEAK and GITRL levels were lower in intensive care unit medical patients. Levels of TWEAK were further decreased in septic patients, and alterations in TWEAK concentrations were linked to an unfavorable outcome. Together with recently published results on other TNFR ligands, these data indicate specific functions of the different TNFR ligands in septic diseases.

Elevated Omentin Serum Levels Predict Long-Term Survival in Critically Ill Patients.

Introduction. Omentin, a recently described adipokine, was shown to be involved in the pathophysiology of inflammatory and infectious diseases. However, its role in critical illness and sepsis is currently unknown. Materials and Methods. Omentin serum concentrations were measured in 117 ICU-patients (84 with septic and 33 with nonseptic disease etiology) admitted to the medical ICU. Results were compared with 50 healthy controls. Results. Omentin serum levels of critically ill patients at admission to the ICU or after 72 hours of ICU treatment were similar compared to healthy controls. Moreover, circulating omentin levels were independent of sepsis and etiology of critical illness. Notably, serum concentrations of omentin could not be linked to concentrations of inflammatory cytokines or routinely used sepsis markers. While serum levels of omentin were not predictive for short term survival during ICU treatment, low omentin concentrations were an independent predictor of patients' overall survival. Omentin levels strongly correlated with that of other adipokines (e.g., leptin receptor or adiponectin), which have also been identified as prognostic markers in critical illness. Conclusions. Although circulating omentin levels did not differ between ICU-patients and controls, elevated omentin levels were predictive for an impaired patients' long term survival.

Plasma membrane-associated nucleoside diphosphate kinase (nm23) in the heart is regulated by beta-adrenergic signaling.

1. Receptor-independent activation of heterotrimeric G proteins by plasma membrane-associated nucleoside diphosphate kinase (NDPK) has been demonstrated in vivo, and elevated levels of NDPK were found in purified sarcolemmal membranes of patients with end-stage heart failure. 2. Among 22 consecutive patients with chronic heart failure who underwent cardiac transplantation, those treated with a beta-blocker (n=8) had a 65% lower NDPK content and activity in the cardiac sarcolemma, compared to patients with similar base line characteristics who had no beta-blocker therapy (n=14). 3. The lower NDPK was associated with a reduced NDPK-dependent, Gi-mediated inhibition of adenylyl cyclase activity, as assessed by in vitro measurement of adenylyl cyclase activity in the presence of GDP or its kinase-resistant analog guanosine 5'-O-(2-thio)diphosphate (GDPbetaS). 4. We further tested whether treatment with a beta-adrenergic agonist would induce an increase in sarcolemmal NDPK. Rats treated with isoproterenol developed myocardial hypertrophy, and NDPK in the sarcolemma rose by 60% during 14 days of treatment. The beta-blocker propranolol prevented both effects. When hypertrophy was induced with thyroid hormone, NDPK did not increase. 5. In conclusion, chronic activation of beta-adrenergic receptors increases the binding of NDPK to cardiac sarcolemma, where it may activate heterotrimeric G proteins.

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.

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

ß-adrenoceptors (ßAR) 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 Gßγ dimers thereby activating and stabilizing heterotrimeric G(s) proteins, key transducer of ßAR signals into the cell. Here, we explored the requirement of NDPK B for basal and ßAR-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.

Decreased contractility due to energy deprivation in a transgenic rat model of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is associated with cardiac hypertrophy, diastolic dysfunction, and sudden death. Recently, it has been suggested that inefficient energy utilization could be a common molecular pathway of HCM-related mutations. We have previously generated transgenic Sprague-Dawley rats overexpressing a truncated cardiac troponin T (DEL-TNT) molecule, displaying typical features of HCM such as diastolic dysfunction and an increased susceptibility to ventricular arrhythmias. We now studied these rats using 31P magnetic resonance spectroscopy (MRS). MRS demonstrated that cardiac energy metabolism was markedly impaired, as indicated by a decreased phosphocreatine to ATP ratio (-31%, p < 0.05). In addition, we assessed contractility of isolated cardiomyocytes. While DEL-TNT and control cardiomyocytes showed no difference under baseline conditions, DEL-TNT cardiomyocytes selectively exhibited a decrease in fractional shortening by 28% after 1 h in glucose-deprived medium (p < 0.05). Moreover, significant decreases in contraction velocity and relaxation velocity were observed. To identify the underlying molecular pathways, we performed transcriptional profiling using real-time PCR. DEL-TNT hearts exhibited induction of several genes critical for cardiac energy supply, including CD36, CPT-1/-2, and PGC-1alpha. Finally, DEL-TNT rats and controls were studied by radiotelemetry after being stressed by isoproterenol, revealing a significantly increased frequency of arrhythmias in transgenic animals. In summary, we demonstrate profound energetic alterations in DEL-TNT hearts, supporting the notion that inefficient cellular ATP utilization contributes to the pathogenesis of HCM.

High energy phosphate transfer by NDPK B/Gbetagammacomplexes--an alternative signaling pathway involved in the regulation of basal cAMP production.

The activation of heterotrimeric G proteins induced by G protein coupled receptors (GPCR) is generally believed to occur by a GDP/GTP exchange at the G protein alpha -subunit. Nevertheless, nucleoside diphosphate kinase (NDPK) and the beta-subunit of G proteins (Gbeta) participate in G protein activation by phosphate transfer reactions leading to the formation of GTP from GDP. Recent work elucidated the role of these reactions. Apparently, the NDPK isoform B (NDPK B) forms a complex with Gbetagamma dimers in which NDPK B acts as a histidine kinase phosphorylating Gbeta at His266. Out of this high energetic phosphoamidate bond the phosphate can be transferred specifically onto GDP. The formed GTP binds to the G protein alpha-subunit and thus activates the respective G protein. Evidence is presented, that this process occurs independent of the classical GPCR-induced GTP/GTP exchange und thus contributes, e.g. to the regulation of basal cAMP synthesis in cells.