Publications by authors named "Kristina Lorenz"

59 Publications

Sulforaphane exposure impairs contractility and mitochondrial function in three-dimensional engineered heart tissue.

Redox Biol 2021 Mar 31;41:101951. Epub 2021 Mar 31.

Institute of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany; DZHK (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany. Electronic address:

Sulforaphane (SFN) is a phytochemical compound extracted from cruciferous plants, like broccoli or cauliflower. Its isothiocyanate group renders SFN reactive, thus allowing post-translational modification of cellular proteins to regulate their function with the potential for biological and therapeutic actions. SFN and stabilized variants recently received regulatory approval for clinical studies in humans for the treatment of neurological disorders and cancer. Potential unwanted side effects of SFN on heart function have not been investigated yet. The present study characterizes the impact of SFN on cardiomyocyte contractile function in cardiac preparations from neonatal rat, adult mouse and human induced-pluripotent stem cell-derived cardiomyocytes. This revealed a SFN-mediated negative inotropic effect, when administered either acutely or chronically, with an impairment of the Frank-Starling response to stretch activation. A direct effect of SFN on myofilament function was excluded in chemically permeabilized mouse trabeculae. However, SFN pretreatment increased lactate formation and enhanced the mitochondrial production of reactive oxygen species accompanied by a significant reduction in the mitochondrial membrane potential. Transmission electron microscopy revealed disturbed sarcomeric organization and inflated mitochondria with whorled membrane shape in response to SFN exposure. Interestingly, administration of the alternative energy source l-glutamine to the medium that bypasses the uptake route of pyruvate into the mitochondrial tricarboxylic acid cycle improved force development in SFN-treated EHTs, suggesting indeed mitochondrial dysfunction as a contributor of SFN-mediated contractile dysfunction. Taken together, the data from the present study suggest that SFN might impact negatively on cardiac contractility in patients with cardiovascular co-morbidities undergoing SFN supplementation therapy. Therefore, cardiac function should be monitored regularly to avoid the onset of cardiotoxic side effects.
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http://dx.doi.org/10.1016/j.redox.2021.101951DOI Listing
March 2021

Simple Targeted Assays for Metabolic Pathways and Signaling: A Powerful Tool for Targeted Proteomics.

Anal Chem 2020 10 21;92(20):13672-13676. Epub 2020 Sep 21.

Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Otto-Hahn-Strasse 6b, 44227 Dortmund, Germany.

We introduce STAMPS, a pathway-centric web service for the development of targeted proteomics assays. STAMPS guides the user by providing several intuitive interfaces for a rapid and simplified method design. Applying our curated framework to signaling and metabolic pathways, we reduced the average assay development time by a factor of ∼150 and revealed that the insulin signaling is actively controlled by protein abundance changes in insulin-sensitive and -resistance states. Although at the current state STAMPS primarily contains mouse data, it was designed for easy extension with additional organisms.
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http://dx.doi.org/10.1021/acs.analchem.0c02793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586293PMC
October 2020

Modeling atrial fibrosis in vitro-Generation and characterization of a novel human atrial fibroblast cell line.

FEBS Open Bio 2020 07 2;10(7):1210-1218. Epub 2020 Jun 2.

Institute of Pharmacology and Toxicology, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Atrial fibrillation (AF) is regularly accompanied by cardiac fibrosis and concomitant heart failure. Due to the heterogeneous nature and complexity of fibrosis, the knowledge about the underlying mechanisms is limited, which prevents effective pharmacotherapy. A deeper understanding of cardiac fibroblasts is essential to meet this need. We previously described phenotypic and functional differences between atrial fibroblasts from patients in sinus rhythm and with AF. Herein, we established and characterized a novel human atrial fibroblast line, which displays typical fibroblast morphology and function comparable to primary cells but with improved proliferation capacity and low spontaneous myofibroblast differentiation. These traits make our model suitable for the study of fibrosis mechanisms and for drug screening aimed at developing effective antifibrotic pharmacotherapy.
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http://dx.doi.org/10.1002/2211-5463.12896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327914PMC
July 2020

Interference with ERK-dimerization at the nucleocytosolic interface targets pathological ERK1/2 signaling without cardiotoxic side-effects.

Nat Commun 2020 04 7;11(1):1733. Epub 2020 Apr 7.

Institute of Pharmacology and Toxicology, University of Würzburg, 97078, Würzburg, Germany.

Dysregulation of extracellular signal-regulated kinases (ERK1/2) is linked to several diseases including heart failure, genetic syndromes and cancer. Inhibition of ERK1/2, however, can cause severe cardiac side-effects, precluding its wide therapeutic application. ERK-autophosphorylation was identified to cause pathological cardiac hypertrophy. Here we report that interference with ERK-dimerization, a prerequisite for ERK-phosphorylation, minimizes cardiac hypertrophy without inducing cardiac adverse effects: an ERK-dimerization inhibitory peptide (EDI) prevents ERK-phosphorylation, nuclear ERK1/2-signaling and cardiomyocyte hypertrophy, protecting from pressure-overload-induced heart failure in mice whilst preserving ERK1/2-activity and cytosolic survival signaling. We also examine this alternative ERK1/2-targeting strategy in cancer: indeed, ERK-phosphorylation is strongly upregulated in cancer and EDI efficiently suppresses cancer cell proliferation without causing cardiotoxicity. This powerful cardio-safe strategy of interfering with ERK-dimerization thus combats pathological ERK1/2-signaling in heart and cancer, and may potentially expand therapeutic options for ERK1/2-related diseases, such as heart failure and genetic syndromes.
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http://dx.doi.org/10.1038/s41467-020-15505-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138859PMC
April 2020

The N-termini of GRK2 and GRK3 simulate the stimulating effects of RKIP on β-adrenoceptors.

Biochem Biophys Res Commun 2019 12 8;520(2):327-332. Epub 2019 Oct 8.

Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Bunsen-Kirchhoff-Str. 11, 44139, Dortmund, Germany; Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Str. 9, 97078, Würzburg, Germany; Comprehensive Heart Failure Center, University Hospital of Würzburg, Am Schwarzenberg 15, 97078, Würzburg, Germany. Electronic address:

The Raf kinase inhibitor protein (RKIP) activates β-adrenoceptors (β-AR) and thereby induces a well-tolerated cardiac contractility and prevents heart failure in mice. Different to RKIP-mediated β-AR activation, chronic activation of β-AR by catecholamines was shown to be detrimental for the heart. RKIP is an endogenous inhibitor of G protein coupled receptor kinase 2 (GRK2); it binds GRK2 and thereby inhibits GRK2 mediated β-AR phosphorylation and desensitization. Here, we evaluate RKIP-mediated effects on β-AR to explore new strategies for β-AR modulation. Co-immunoprecipitation assays and pull-down assays revealed subtype specificity of RKIP for the cardiac GRK isoforms GRK2 and GRK3 - not GRK5 - as well as several RKIP binding sites within their N-termini (GRK2 and GRK3). Overexpression of these N-termini prevented β-AR phosphorylation and internalization, subsequently increased receptor signaling in HEK293 cells and cardiomyocyte contractility. Co-immunoprecipitation assays of β-AR with these N-terminal GRK fragments revealed a direct interaction suggesting a steric interference of the fragments with the functional GRK-receptor interaction. Altogether, N-termini of GRK2 and GRK3 efficiently simulate RKIP effects on β-AR signaling in HEK293 cells and in cardiomyocytes by their binding to β-AR and, thus, provide important insights for the development of new strategies to modulate β-AR signaling.
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http://dx.doi.org/10.1016/j.bbrc.2019.09.135DOI Listing
December 2019

Assessing the role of extracellular signal-regulated kinases 1 and 2 in volume overload-induced cardiac remodelling.

ESC Heart Fail 2019 10 19;6(5):1015-1026. Epub 2019 Jul 19.

Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany.

Aims: Volume overload (VO) and pressure overload (PO) induce differential cardiac remodelling responses including distinct signalling pathways. Extracellular signal-regulated kinases 1 and 2 (ERK1/2), key signalling components in the mitogen-activated protein kinase (MAPK) pathways, modulate cardiac remodelling during pressure overload (PO). This study aimed to assess their role in VO-induced cardiac remodelling as this was unknown.

Methods And Results: Aortocaval fistula (Shunt) surgery was performed in mice to induce cardiac VO. Two weeks of Shunt caused a significant reduction of cardiac ERK1/2 activation in wild type (WT) mice as indicated by decreased phosphorylation of the TEY (Thr-Glu-Tyr) motif (-28% as compared with Sham controls, P < 0.05). Phosphorylation of other MAPKs was unaffected. For further assessment, transgenic mice with cardiomyocyte-specific ERK2 overexpression (ERK2tg) were studied. At baseline, cardiac ERK1/2 phosphorylation in ERK2tg mice remained unchanged compared with WT littermates, and no overt cardiac phenotype was observed; however, cardiac expression of the atrial natriuretic peptide was increased on messenger RNA (3.6-fold, P < 0.05) and protein level (3.1-fold, P < 0.05). Following Shunt, left ventricular dilation and hypertrophy were similar in ERK2tg mice and WT littermates. Left ventricular function was maintained, and changes in gene expression indicated reactivation of the foetal gene program in both genotypes. No differences in cardiac fibrosis and kinase activation was found amongst all experimental groups, whereas apoptosis was similarly increased through Shunt in ERK2tg and WT mice.

Conclusions: VO-induced eccentric hypertrophy is associated with reduced cardiac ERK1/2 activation in vivo. Cardiomyocyte-specific overexpression of ERK2, however, does not alter cardiac remodelling during VO. Future studies need to define the pathophysiological relevance of decreased ERK1/2 signalling during VO.
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http://dx.doi.org/10.1002/ehf2.12497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816056PMC
October 2019

How to Steer and Control ERK and the ERK Signaling Cascade Exemplified by Looking at Cardiac Insufficiency.

Int J Mol Sci 2019 May 2;20(9). Epub 2019 May 2.

Biozentrum, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany.

Mathematical optimization framework allows the identification of certain nodes within a signaling network. In this work, we analyzed the complex extracellular-signal-regulated kinase 1 and 2 (ERK1/2) cascade in cardiomyocytes using the framework to find efficient adjustment screws for this cascade that is important for cardiomyocyte survival and maladaptive heart muscle growth. We modeled optimal pharmacological intervention points that are beneficial for the heart, but avoid the occurrence of a maladaptive ERK1/2 modification, the autophosphorylation of ERK at threonine 188 (ERK Thr 188 phosphorylation), which causes cardiac hypertrophy. For this purpose, a network of a cardiomyocyte that was fitted to experimental data was equipped with external stimuli that model the pharmacological intervention points. Specifically, two situations were considered. In the first one, the cardiomyocyte was driven to a desired expression level with different treatment strategies. These strategies were quantified with respect to beneficial effects and maleficent side effects and then which one is the best treatment strategy was evaluated. In the second situation, it was shown how to model constitutively activated pathways and how to identify drug targets to obtain a desired activity level that is associated with a healthy state and in contrast to the maleficent expression pattern caused by the constitutively activated pathway. An implementation of the algorithms used for the calculations is also presented in this paper, which simplifies the application of the presented framework for drug targeting, optimal drug combinations and the systematic and automatic search for pharmacological intervention points. The codes were designed such that they can be combined with any mathematical model given by ordinary differential equations.
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http://dx.doi.org/10.3390/ijms20092179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6539830PMC
May 2019

Analysis of fibrosis in control or pressure overloaded rat hearts after mechanical unloading by heterotopic heart transplantation.

Sci Rep 2019 04 5;9(1):5710. Epub 2019 Apr 5.

DZHK (German Centre for Cardiovascular Research) partner site Hamburg/Kiel/Lübeck, Hamburg, Germany.

Mechanical unloading (MU) by implantation of left ventricular assist devices (LVAD) has become clinical routine. This procedure has been shown to reverse cardiac pathological remodeling, with the underlying molecular mechanisms incompletely understood. Most studies thus far were performed in non-standardized human specimens or MU of healthy animal hearts. Our study investigates cardiac remodeling processes in sham-operated healthy rat hearts and in hearts subjected to standardized pathological pressure overload by transverse aortic constriction (TAC) prior to MU by heterotopic heart transplantation (hHTx/MU). Rats underwent sham or TAC surgery. Disease progression was monitored by echocardiography prior to MU by hHTx/MU. Hearts after TAC or TAC combined with hHTx/MU were removed and analyzed by histology, western immunoblot and gene expression analysis. TAC surgery resulted in cardiac hypertrophy and impaired cardiac function. TAC hearts revealed significantly increased cardiac myocyte diameter and mild fibrosis. Expression of hypertrophy associated genes after TAC was higher compared to hearts after hHTx/MU. While cardiac myocyte cell diameter regressed to the level of sham-operated controls in all hearts subjected to hHTx/MU, fibrotic remodeling was significantly exacerbated. Transcription of pro-fibrotic and apoptosis-related genes was markedly augmented in all hearts after hHTx/MU. Sarcomeric proteins involved in excitation-contraction coupling displayed significantly lower phosphorylation levels after TAC and significantly reduced total protein levels after hHTx/MU. Development of myocardial fibrosis, cardiac myocyte atrophy and loss of sarcomeric proteins was observed in all hearts that underwent hHTX/MU regardless of the disease state. These results may help to explain the clinical experience with low rates of LVAD removal due to lack of myocardial recovery.
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http://dx.doi.org/10.1038/s41598-019-42263-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451012PMC
April 2019

Association between Comorbidities and Progression of Transvalvular Pressure Gradients in Patients with Moderate and Severe Aortic Valve Stenosis.

Cardiol Res Pract 2018 11;2018:3713897. Epub 2018 Nov 11.

Department of Internal Medicine I, University Hospital Würzburg, Würzburg, Germany.

Background: Fast progression of the transaortic mean gradient () is relevant for clinical decision making of valve replacement in patients with moderate and severe aortic stenosis (AS) patients. However, there is currently little knowledge regarding the determinants affecting progression of transvalvular gradient in AS patients.

Methods: This monocentric retrospective study included consecutive patients presenting with at least two transthoracic echocardiography examinations covering a time interval of one year or more between April 2006 and February 2016 and diagnosed as moderate or severe aortic stenosis at the final echocardiographic examination. Laboratory parameters, medication, and prevalence of eight known cardiac comorbidities and risk factors (hypertension, diabetes, coronary heart disease, peripheral artery occlusive disease, cerebrovascular disease, renal dysfunction, body mass index ≥30 Kg/m, and history of smoking) were analyzed. Patients were divided into slow ( < 5 mmHg/year) or fast ( ≥ 5 mmHg/year) progression groups.

Results: A total of 402 patients (mean age 78 ± 9.4 years, 58% males) were included in the study. Mean follow-up duration was 3.4 ± 1.9 years. The average number of cardiac comorbidities and risk factors was 3.1 ± 1.6. Average number of cardiac comorbidities and risk factors was higher in patients in slow progression group than in fast progression group (3.3 ± 1.5 vs 2.9 ± 1.7; =0.036). Patients in slow progression group had more often coronary heart disease (49.2% vs 33.6%; =0.003) compared to patients in fast progression group. LDL-cholesterol values were lower in the slow progression group (100 ± 32.6 mg/dl vs 110.8 ± 36.6 mg/dl; =0.005).

Conclusion: These findings suggest that disease progression of aortic valve stenosis is faster in patients with fewer cardiac comorbidities and risk factors, especially if they do not have coronary heart disease. Further prospective studies are warranted to investigate the outcome of patients with slow versus fast progression of transvalvular gradient with regards to comorbidities and risk factors.
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http://dx.doi.org/10.1155/2018/3713897DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6252229PMC
November 2018

Oral Chaperone Therapy Migalastat for Treating Fabry Disease: Enzymatic Response and Serum Biomarker Changes After 1 Year.

Clin Pharmacol Ther 2019 05 13;105(5):1224-1233. Epub 2019 Jan 13.

Department of Internal Medicine I and Comprehensive Heart Failure Center, University Hospital Würzburg, Würzburg, Germany.

Long-term effects of migalastat therapy in clinical practice are currently unknown. We evaluated migalastat efficacy and biomarker changes in a prospective, single-center study on 14 patients with Fabry disease (55 ± 14 years; 11 men). After 1 year of open-label migalastat therapy, patients showed significant changes in alpha-galactosidase-A activity (0.06-0.2 nmol/minute/mg protein; P = 0.001), left ventricular myocardial mass index (137-130 g/m ; P = 0.037), and serum creatinine (0.94-1.0 mg/dL; P = 0.021), accounting for deterioration in estimated glomerular filtration rate (87-78 mL/minute/1.73 m ; P = 0.012). The enzymatic increase correlated with myocardial mass reduction (r = -0.546; P = 0.044) but not with renal function (r = -0.086; P = 0.770). Plasma globotriaosylsphingosine was reduced in therapy-naive patients (10.9-6.0 ng/mL; P = 0.021) and stable (9.6-12.1 ng/mL; P = 0.607) in patients switched from prior enzyme-replacement therapy. These first real-world data show that migalastat substantially increases alpha-galactosidase-A activity, stabilizes related serum biomarkers, and improves cardiac integrity in male and female patients with amenable Fabry disease mutations.
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http://dx.doi.org/10.1002/cpt.1321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590383PMC
May 2019

Real-time Triggered RAdial Single-Shot Inversion recovery for arrhythmia-insensitive myocardial T1 mapping: motion phantom validation and in vivo comparison.

Magn Reson Med 2019 03 12;81(3):1714-1725. Epub 2018 Nov 12.

Comprehensive Heart Failure Center (CHFC), University Hospital Würzburg, Würzburg, Germany.

Purpose: Cardiac T mapping has become an increasingly important imaging technique, contributing novel diagnostic options. However, currently utilized methods are often associated with accuracy problems because of heart rate variations and cardiac arrhythmia, limiting their value in clinical routine. This study aimed to introduce an improved arrhythmia-related robust T mapping sequence called RT-TRASSI (real-time Triggered RAdial Single-Shot Inversion recovery).

Methods: All measurements were performed on a 3.0T whole-body imaging system. A real-time feedback algorithm for arrhythmia detection was implemented into the previously described pulse sequence. A programmable motion phantom was constructed and measurements with different simulated arrhythmias arranged. T mapping accuracy and susceptibility to artifacts were analyzed. In addition, in vivo measurements and comparisons with 3 prevailing T mapping sequences (MOLLI, ShMOLLI, and SASHA) were carried out to investigate the occurrence of artifacts.

Results: In the motion phantom measurements, RT-TRASSI showed excellent agreement with predetermined reference T values. Percentage scattering of the T values ranged from -0.6% to +1.9% in sinus rhythm and -1.0% to +3.1% for high-grade arrhythmias. In vivo, RT-TRASSI showed diagnostic image quality with only 6% of the acquired T maps including image artifacts. In contrast, more than 40% of the T maps acquired with MOLLI, ShMOLLI, or SASHA included motion artifacts.

Conclusion: Accuracy issues because of heart rate variability and arrhythmia are a prevailing problem in current cardiac T mapping techniques. With RT-TRASSI, artifacts can be minimized because of the short acquisition time and effective real-time feedback, avoiding potential data acquisition during systolic heart phase.
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http://dx.doi.org/10.1002/mrm.27526DOI Listing
March 2019

Myocardial Fibrosis Predicts 10-Year Survival in Patients Undergoing Aortic Valve Replacement.

Circ Cardiovasc Imaging 2018 08;11(8):e007131

Department of Internal Medicine I, Center of Cardiovascular Diseases, University Hospital, Universitätsklinikum Würzburg, Würzburg, Bayern, Germany (S.H., B.F., T.S., D.L., K.H., D.G., M.C., S.S., W.V., S.F., G.E., P.N.).

Background Long-term data on evolution and clinical impact of myocardial fibrosis in valvular heart disease are scarce. Methods and Results In this 10 years' extension of a prospective study in patients undergoing conventional aortic valve replacement because of symptomatic severe aortic valve stenosis, the impact of myocardial replacement fibrosis (MRF) on long-term outcome was assessed. Endomyocardial biopsies were acquired during aortic valve replacement in 58 consecutive patients. MRF was graded using the calculated percentage area of fibrosis and patients categorized as severe (n=21), mild (n=15), and no fibrosis (n=22). Echocardiography including strain imaging, as well as cardiovascular magnetic resonance, to assess late gadolinium enhancement was performed at baseline, 1, and 10 years after aortic valve replacement. Death of any cause occurred in 21 patients (38.9%): 3 (14.3%) in the group without MRF, 6 (42.9%) in the mild MRF group, and 12 (63.2%) in the severe MRF group ( P=0.006), resulting in the lowest cumulative survival for patients with severe MRF (log-rank P=0.003). In the group without MRF, none died of cardiovascular cause. MRF was found to be an independent predictor of survival (hazard ratio, 1.271; 95% CI, 1.032-1.564; P=0.024). Conclusions This 10-year follow-up study underlines the profound impact of replacement fibrosis with regard to cardiac and all-cause mortality in patients undergoing aortic valve replacement for severe aortic valve stenosis. Integrating cardiovascular magnetic resonance and echocardiographic functional imaging beyond ejection fraction quantification could help in clinical decision making to stratify patient prognosis with regard to myocardial longitudinal function and prevalence of replacement fibrosis.
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http://dx.doi.org/10.1161/CIRCIMAGING.117.007131DOI Listing
August 2018

The A2B adenosine receptor in MDA-MB-231 breast cancer cells diminishes ERK1/2 phosphorylation by activation of MAPK-phosphatase-1.

PLoS One 2018 29;13(8):e0202914. Epub 2018 Aug 29.

Institut für Pharmakologie und Toxikologie, Universität Würzburg, Würzburg, Germany.

It was previously shown that the estrogen-receptor negative breast cancer cell line MBA-MD-231 expresses high levels of A2B adenosine receptors as the sole adenosine receptor subtype. These receptors couple to both, stimulation of adenylyl cyclase and a Ca2+ signal. In order to establish a potential role of A2B adenosine receptors in tumor growth and development MAPK signaling was investigated in these breast cancer cells. Although it is known that A2B adenosine receptors may stimulate MAPK it was found that in MBA-MD-231 cells ERK1/2 phosphorylation is reduced upon agonist-stimulation of A2B adenosine receptors. This reduction is also triggered by forskolin, but abolished by the PKA inhibitor H89, suggesting an important role for the cAMP-PKA pathway. Likewise, a role for intracellular Ca2+ was established as the Ca2+ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) abolished the reduction of ERK1/2 phosphorylation triggered by A2B stimulation. It was shown that various pathways downstream from A2B adenosine receptors resulted in a stimulation of MAPK phosphatase-1 (MKP-1) which dephosphorylates phospho ERK1/2, and thus plays a critical role in the regulation of the phosphorylation state of ERK1/2. The reduction of ERK1/2 phosphorylation mediated by A2B adenosine receptors might provide an interesting approach for adjuvant treatment leading to reduced growth of certain tumors expressing the A2B subtype.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202914PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6114864PMC
February 2019

Biochemical and pathological changes result from mutated Caveolin-3 in muscle.

Skelet Muscle 2018 08 28;8(1):28. Epub 2018 Aug 28.

Biomedical Research Department, Tissue Omics group, Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V, Otto-Hahn-Str. 6b, 44227, Dortmund, Germany.

Background: Caveolin-3 (CAV3) is a muscle-specific protein localized to the sarcolemma. It was suggested that CAV3 is involved in the connection between the extracellular matrix (ECM) and the cytoskeleton. Caveolinopathies often go along with increased CK levels indicative of sarcolemmal damage. So far, more than 40 dominant pathogenic mutations have been described leading to several phenotypes many of which are associated with a mis-localization of the mutant protein to the Golgi. Golgi retention and endoplasmic reticulum (ER) stress has been demonstrated for the CAV3 p.P104L mutation, but further downstream pathophysiological consequences remained elusive so far.

Methods: We utilized a transgenic (p.P104L mutant) mouse model and performed proteomic profiling along with immunoprecipitation, immunofluorescence and immunoblot examinations (including examination of α-dystroglycan glycosylation), and morphological studies (electron and coherent anti-Stokes Raman scattering (CARS) microscopy) in a systematic investigation of molecular and subcellular events in p.P104L caveolinopathy.

Results: Our electron and CARS microscopic as well as immunological studies revealed Golgi and ER proliferations along with a build-up of protein aggregates further characterized by immunoprecipitation and subsequent mass spectrometry. Molecular characterization these aggregates showed affection of mitochondrial and cytoskeletal proteins which accords with our ultra-structural findings. Additional global proteomic profiling revealed vulnerability of 120 proteins in diseased quadriceps muscle supporting our previous findings and providing more general insights into the underlying pathophysiology. Moreover, our data suggested that further DGC components are altered by the perturbed protein processing machinery but are not prone to form aggregates whereas other sarcolemmal proteins are ubiquitinated or bind to p62. Although the architecture of the ER and Golgi as organelles of protein glycosylation are altered, the glycosylation of α-dystroglycan presented unchanged.

Conclusions: Our combined data classify the p.P104 caveolinopathy as an ER-Golgi disorder impairing proper protein processing and leading to aggregate formation pertaining proteins important for mitochondrial function, cytoskeleton, ECM remodeling and sarcolemmal integrity. Glycosylation of sarcolemmal proteins seems to be normal. The new pathophysiological insights might be of relevance for the development of therapeutic strategies for caveolinopathy patients targeting improved protein folding capacity.
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http://dx.doi.org/10.1186/s13395-018-0173-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6114045PMC
August 2018

Oxidation of cardiac myofilament proteins: Priming for dysfunction?

Mol Aspects Med 2018 10 23;63:47-58. Epub 2018 Aug 23.

King's British Heart Foundation Centre, King's College London, UK.

Oxidants are produced endogenously and can react with and thereby post-translationally modify target proteins. They have been implicated in the redox regulation of signal transduction pathways conferring protection, but also in mediating oxidative stress and causing damage. The difference is that in scenarios of injury the amount of oxidants generated is higher and/or the duration of oxidant exposure sustained. In the cardiovascular system, oxidants are important for blood pressure homeostasis, for unperturbed cardiac function and also contribute to the observed protection during ischemic preconditioning. In contrast, oxidative stress accompanies all major cardiovascular pathologies and has been attributed to mediate contractile dysfunction in part by inducing oxidative modifications in myofilament proteins. However, the proportion to which oxidative modifications of contractile proteins are beneficial or causatively mediate disease progression needs to be carefully reconsidered. These antithetical aspects will be discussed in this review with special focus on direct oxidative post-translational modifications of myofilament proteins that have been described to occur in vivo and to regulate actin-myosin interactions in the cardiac myocyte sarcomere, the methodologies for detection of oxidative post-translational modifications in target proteins and the feasibility of antioxidant therapy strategies as a potential treatment for cardiac disorders.
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http://dx.doi.org/10.1016/j.mam.2018.08.003DOI Listing
October 2018

The β agonist terbutaline specifically decreases pulmonary arterial pressure under normoxia and hypoxia via α adrenoceptor antagonism.

FASEB J 2018 05 2;32(5):2519-2530. Epub 2018 Jan 2.

Life and Brain Center, Institute of Physiology I, Medical Faculty, University of Bonn, Bonn, Germany.

Pulmonary hypertension is a severe, incurable disease with a poor prognosis. Although treatment regimens have improved during the last 2 decades, current pharmacologic strategies are limited and focus on the modulation of only a few pathways related to endothelin, NO, and prostacyclin signaling. Therefore, the identification of novel molecular targets is urgently needed. We found that the β adrenoceptor (AR) agonists terbutaline (TER) and salbutamol induced a dose-dependent vasorelaxation in large pulmonary arteries but not aortas of mouse. This effect was found to be independent of β ARs and the endothelium but was determined by the type of the preconstrictor. Vasodilation by β AR agonists occurred after pretreatment of pulmonary arteries with phenylephrine and serotonin, both agonists of α ARs, but was absent after preconstriction with the thromboxane analog U46619. These data indicated α-adrenolytic activity of β AR agonists, which was confirmed by a right shift of the phenylephrine dose-response curve by TER. This effect was physiologically relevant because TER also relaxed small intrapulmonary arteries in lung slices and diminished pulmonary arterial pressure in an isolated perfused lung model under normoxia and hypoxia. Finally, TER applied as an aerosol also selectively decreased pulmonary arterial pressure without effects on systemic blood pressure and heart rate in mouse in vivo. Thus, β AR agonists display α-adrenolytic activity in pulmonary arteries ex vivo and in vivo, and may provide a novel option to reduce pulmonary arterial pressure in pulmonary hypertension.-Neumann, V., Knies, R., Seidinger, A., Simon, A., Lorenz, K., Matthey, M., Breuer, J., Wenzel, D. The β agonist terbutaline specifically decreases pulmonary arterial pressure under normoxia and hypoxia via α adrenoceptor antagonism.
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http://dx.doi.org/10.1096/fj.201700684RRDOI Listing
May 2018

Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome.

Proc Natl Acad Sci U S A 2017 12 5;114(51):13453-13458. Epub 2017 Dec 5.

Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637;

Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or "theft" mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein-coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein-Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.
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http://dx.doi.org/10.1073/pnas.1711543114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754776PMC
December 2017

α-Galactosidase A Genotype N215S Induces a Specific Cardiac Variant of Fabry Disease.

Circ Cardiovasc Genet 2017 Oct;10(5)

From the Department of Internal Medicine I and Comprehensive Heart Failure Center (CHFC) (D.O., D.L., K.H., T.S., J.M., K.L., G.E., C.W., P.N.), Fabry Center for Interdisciplinary Therapy (FAZIT) (D.O., D.L., K.H., N.Ü., T.S., J.M., C.S., G.E., C.W., P.N.), and Department of Neurology (N.Ü., C.S.), University Hospital Würzburg, Germany; West German Heart and Vascular Center Essen, University Hospital Essen, Germany (K.L.); Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany (K.L.); Department of Molecular Pathology, University Hospital of Tübingen, Germany (R.K.); and Department of Cellular and Molecular Pathology, German Cancer Research Center (DKFZ), Heidelberg, Germany (H.-J.G.).

Background: Hypertrophic cardiomyopathy is the most common type of cardiomyopathy, but many patients lack sarcomeric/myofilament mutations. We studied whether cardio-specific α-galactosidase A gene variants are misinterpreted as hypertrophic cardiomyopathy because of the lack of extracardiac organ involvement.

Methods And Results: All subjects who tested positive for the N215S genotype (n=26, 13 females, mean age 49±17 [range, 14-74] years) were characterized in this prospective monocentric longitudinal cohort study to determine genotype-specific clinical characteristics of the N215S (c.644A>G [p.Asn215Ser]) α-galactosidase A gene variant. All subjects were initially referred with suspicion of genetically determined hypertrophic cardiomyopathy. Cardiac hypertrophy (interventricular septum, 12±4 [7-23] mm; left ventricular posterior wall, 11±4 [7-21] mm; left ventricular mass, 86±41 [46-195] g/m) was progressive, systolic function mainly preserved (cardiac index 2.8±0.6 [1.9-3.9] L/min per m), and diastolic function mildly abnormal. Cardiac magnetic resonance imaging revealed replacement fibrosis in (18/26, 69%), particularly in subjects >50 years. Elderly subjects had advanced heart failure, and 6 (23%) were suggested for implantable cardioverter-defibrillator therapy. Leukocyte α-galactosidase A enzyme activity was mildly reduced in 19 subjects and lyso-globotriaosylceramide slightly elevated (median, 4.9; interquartile range, 1.3-9.1 ng/mL). Neurological and renal impairments (serum creatinine, 0.87±0.20; median, 0.80; interquartile range, 0.70-1.01 mg/dL; glomerular filtration rate, 102±23; median, 106; interquartile range, 84-113 mL/min) were discreet. Only 2 subjects developed clinically relevant proteinuria.

Conclusions: α-Galactosidase A genotype N215S does not lead to the development of a classical Fabry phenotype but induces a specific cardiac variant of Fabry disease mimicking nonobstructive hypertrophic cardiomyopathy. The lack of prominent noncardiac impairment leads to a significant delay in diagnosis and Fabry-specific therapy.
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http://dx.doi.org/10.1161/CIRCGENETICS.116.001691DOI Listing
October 2017

Raf kinase inhibitor protein: lessons of a better way for β-adrenergic receptor activation in the heart.

J Physiol 2017 06 23;595(12):4073-4087. Epub 2017 May 23.

Institute of Pharmacology and Clinical Pharmacology, Düsseldorf University Hospital, Universtitätsstraße 1, 40225, Düsseldorf, Germany.

Stimulation of β-adrenergic receptors (βARs) provides the most efficient physiological mechanism to enhance contraction and relaxation of the heart. Activation of βARs allows rapid enhancement of myocardial function in order to fuel the muscles for running and fighting in a fight-or-flight response. Likewise, βARs become activated during cardiovascular disease in an attempt to counteract the restrictions of cardiac output. However, long-term stimulation of βARs increases the likelihood of cardiac arrhythmias, adverse ventricular remodelling, decline of cardiac performance and premature death, thereby limiting the use of βAR agonists in the treatment of heart failure. Recently the endogenous Raf kinase inhibitor protein (RKIP) was found to activate βAR signalling of the heart without adverse effects. This review will summarize the current knowledge on RKIP-driven compared to receptor-mediated signalling in cardiomyocytes. Emphasis is given to the differential effects of RKIP on β - and β -ARs and their downstream targets, the regulation of myocyte calcium cycling and myofilament activity.
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http://dx.doi.org/10.1113/JP274064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5471367PMC
June 2017

Sex-difference in expression and function of beta-adrenoceptors in macrovessels: role of the endothelium.

Basic Res Cardiol 2017 05 7;112(3):29. Epub 2017 Apr 7.

Department of Physiology, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstr. 74, 01307, Dresden, Germany.

Estrogen modulates adrenergic reactivity of macrovessels, resulting in weaker α-adrenergic vasoconstriction in females than males. However, the mechanisms governing this important sex-specific difference are not well understood. We hypothesized that vessels of females express more dilatory β-adrenoceptors, which counteract constrictive effects of α-adrenoceptors. This hypothesis was tested using aortas of normotensive (WKY) and hypertensive rats (SHR), along with human mammary artery. Selective blockade of β (CGP20712) or β (SR59230A), but not β (ICI118,551) adrenoceptors, greatly increased α-adrenergic constriction (norepinephrine) of aorta in female SHRs, but not in male SHRs at 12 weeks of age. Consistently, the selective β/β (isoproterenol) and β-adrenergic (BRL37344) relaxation was stronger in female SHRs than in males. Removal of endothelium and use of L-NMMA abolished sex-difference in α-adrenergic constriction and β-adrenergic relaxation. Immunostainings revealed endothelial localization of β- and β-adrenoceptors. mRNA levels of aortic β- and β-, but not β-adrenoceptors were markedly higher in female than in male SHRs. The sex-specific differences in α-adrenergic constriction and β-adrenoceptor mRNA levels were age-dependent, predominantly present up to 29 weeks and disappeared at 36 weeks of age. The sex-specific difference was not strain-dependent and was similarly present in normotensive WKY rats. Human mammary artery of women showed a weaker α-adrenergic constriction than arteries of men. This sex-specific difference was prominent at 45-65 years and disappeared with aging. Our results convincingly demonstrate that female macrovessels express more dilatory β- and β-adrenoreceptors than male vessels with a predominant endothelial localization. This sex-specific difference is functionally relevant in young adults and is attenuated with aging.
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http://dx.doi.org/10.1007/s00395-017-0617-2DOI Listing
May 2017

Cardiac amyloidosis mimicking severe aortic valve stenosis - a case report demonstrating diagnostic pitfalls and role of dobutamine stress echocardiography.

BMC Cardiovasc Disord 2017 03 22;17(1):86. Epub 2017 Mar 22.

Department of Internal Medicine I, University Hospital Würzburg, Würzburg, Germany.

Background: Aortic valve stenosis is a common finding diagnosed with high sensitivity in transthoracic echocardiography, but the examiner often finds himself confronted with uncertain results in patients with moderate pressure gradients and concomitant systolic heart failure. While patients with true-severe low-gradient aortic valve stenosis with either reduced or preserved left ventricular systolic function are primarily candidates for valve replacement, there is a relevant proportion of patients with pseudo-severe aortic valve stenosis anticipated not to benefit but actually rather deteriorate by interventional therapy or surgery.

Case Presentation: In this article we present a case report of a male patient with pseudo-severe aortic valve stenosis due to cardiac amyloidosis highlighting the diagnostic schedule. The patient underwent stress echocardiography because of discrepant findings in transthoracic echocardiography and cardiac catheterization regarding the severity of aortic valve stenosis. After evaluation of the results, it became clear that he had a need for optimum heart failure medication and implantation of a cardiac resynchronization therapy defibrillator.

Conclusion: Due to the pitfalls in conventional as well as invasive diagnostics at rest, Stress echocardiography should be considered part of the standard optimum diagnostic spectrum in all unclear or borderline cases in order to confirm the correct diagnosis and constitute optimal therapy.
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http://dx.doi.org/10.1186/s12872-017-0519-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5361717PMC
March 2017

Multi-OMICS: a critical technical perspective on integrative lipidomics approaches.

Biochim Biophys Acta Mol Cell Biol Lipids 2017 Aug 11;1862(8):808-811. Epub 2017 Feb 11.

Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Otto-Hahn-Str. 6b, Dortmund, Germany. Electronic address:

During the past decades, high-throughput approaches for analyzing different molecular classes such as nucleic acids, proteins, metabolites, and lipids have grown rapidly. These approaches became powerful tools for getting a fundamental understanding of biological systems. Considering each approach and its results separately, relations and causal connections between these classes have no chance to be revealed, since only separate molecular snapshots are provided. Only a combined approach, not fully established yet, with the integration of the corresponding data, might yield a comprehensive and complete understanding of biological processes, such as crosstalk and interactions in signaling pathways. Taking two or more omics-methods into consideration for analysis is referred to as a multi-omics approach, which is gradually evolving. In this critical note, we briefly discuss the relevance, challenges, current state, and potential of data integration from multi-omics approaches, with a special focus on lipidomics analysis, listing the advantages and gaps in this field. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.
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http://dx.doi.org/10.1016/j.bbalip.2017.02.003DOI Listing
August 2017

Eukaryotic elongation factor 2 is a prognostic marker and its kinase a potential therapeutic target in HCC.

Oncotarget 2017 Feb;8(7):11950-11962

Institute of Pathology, University of Duisburg-Essen, Essen, Germany.

Hepatocellular carcinoma is a cancer with increasing incidence and largely refractory to current anticancer drugs. Since Sorafenib, a multikinase inhibitor has shown modest efficacy in advanced hepatocellular carcinoma additional treatments are highly needed. Protein phosphorylation via kinases is an important post-translational modification to regulate cell homeostasis including proliferation and apoptosis. Therefore kinases are valuable targets in cancer therapy. To this end we performed 2D differential gel electrophoresis and mass spectrometry analysis of phosphoprotein-enriched lysates of tumor and corresponding non-tumorous liver samples to detect differentially abundant phosphoproteins to screen for novel kinases as potential drug targets. We identified 34 differentially abundant proteins in phosphoprotein enriched lysates. Expression and distribution of the candidate protein eEF2 and its phosphorylated isoform was validated immunohistochemically on 78 hepatocellular carcinoma and non-tumorous tissue samples. Validation showed that total eEF2 and phosphorylated eEF2 at threonine 56 are prognostic markers for overall survival of HCC-patients. The activity of the regulating eEF2 kinase, compared between tumor and non-tumorous tissue lysates by in vitro kinase assays, is more than four times higher in tumor tissues. Functional analyzes regarding eEF2 kinase were performed in JHH5 cells with CRISPR/Cas9 mediated eEF2 kinase knock out. Proliferation and growth is decreased in eEF2 kinase knock out cells.

Conclusion: eEF2 and phosphorylated eEF2 are prognostic markers for survival of hepatocellular carcinoma patients and the regulating eEF2 kinase is a potential drug target for tumor therapy.
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http://dx.doi.org/10.18632/oncotarget.14447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355317PMC
February 2017

Phosphodiesterase 2 Protects Against Catecholamine-Induced Arrhythmia and Preserves Contractile Function After Myocardial Infarction.

Circ Res 2017 Jan 31;120(1):120-132. Epub 2016 Oct 31.

From the Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Mannheim, Heidelberg University, Germany (C.V., T.W.); Institute of Pharmacology, University Medical Center Göttingen (UMG) Heart Center, Georg August University Medical School Göttingen, Germany (C.V., M.D., M.R., S.M.); UMR-S 1180, INSERM, Université Paris-Sud, Université Paris-Saclay, Châtenay-Malabry, France (M.L., H.M., S.K., P.L., J.L., G.V., R.F.); Department of Molecular Cardiology and Epigenetics, University Hospital Heidelberg, Germany (M.D.); Institute of Pharmacology and Toxicology, University of Würzburg and Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany (K.L., C.S.); Comprehensive Heart Failure Center, University of Würzburg, and West German Heart and Vascular Center Essen, Germany (K.L.); Department of Pharmacology and Toxicology, University of Technology Dresden, Germany (S.L., M.W., A.E.-A.); Department of Cardiology and Pneumology, Center of Molecular Cardiology, UMG Heart Center, Georg August University Medical School Göttingen, Germany (F.E.M., S.S.); Department of Internal Medicine III, Cardiology and Angiology, University of Kiel, Germany (S.S.); BAYER Pharma AG, Wuppertal, Germany (A.G., M.H., F.W.); University Heart Center, Department of General and Interventional Cardiology, University Medical Center Hamburg-Eppendorf, Germany (F.J.B.); and DZHK (German Centre for Cardiovascular Research), partner sites Heidelberg/Mannheim, Göttingen and Hamburg/Kiel/Lübeck, Germany (C.V., M.D., M.R., S.M., F.E.M., S.S., F.J.B., T.W.).

Rationale: Phosphodiesterase 2 is a dual substrate esterase, which has the unique property to be stimulated by cGMP, but primarily hydrolyzes cAMP. Myocardial phosphodiesterase 2 is upregulated in human heart failure, but its role in the heart is unknown.

Objective: To explore the role of phosphodiesterase 2 in cardiac function, propensity to arrhythmia, and myocardial infarction.

Methods And Results: Pharmacological inhibition of phosphodiesterase 2 (BAY 60-7550, BAY) led to a significant positive chronotropic effect on top of maximal β-adrenoceptor activation in healthy mice. Under pathological conditions induced by chronic catecholamine infusions, BAY reversed both the attenuated β-adrenoceptor-mediated inotropy and chronotropy. Conversely, ECG telemetry in heart-specific phosphodiesterase 2-transgenic (TG) mice showed a marked reduction in resting and in maximal heart rate, whereas cardiac output was completely preserved because of greater cardiac contraction. This well-tolerated phenotype persisted in elderly TG with no indications of cardiac pathology or premature death. During arrhythmia provocation induced by catecholamine injections, TG animals were resistant to triggered ventricular arrhythmias. Accordingly, Ca-spark analysis in isolated TG cardiomyocytes revealed remarkably reduced Ca leakage and lower basal phosphorylation levels of Ca-cycling proteins including ryanodine receptor type 2. Moreover, TG demonstrated improved cardiac function after myocardial infarction.

Conclusions: Endogenous phosphodiesterase 2 contributes to heart rate regulation. Greater phosphodiesterase 2 abundance protects against arrhythmias and improves contraction force after severe ischemic insult. Activating myocardial phosphodiesterase 2 may, thus, represent a novel intracellular antiadrenergic therapeutic strategy protecting the heart from arrhythmia and contractile dysfunction.
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http://dx.doi.org/10.1161/CIRCRESAHA.116.310069DOI Listing
January 2017

Coagulation factor XII induces pro-inflammatory cytokine responses in macrophages and promotes atherosclerosis in mice.

Thromb Haemost 2017 01 27;117(1):176-187. Epub 2016 Oct 27.

Dr. Alma Zernecke, Universitätsklinikum Würzburg, Institut für Experimentelle Biomedizin, Josef-Schneider-Str. 2, 97080 Würzburg, Germany, Tel: +49 201 48331, Fax: +49 201 648341, E-mail:

Atherosclerosis is considered a chronic inflammatory disease of the vessel wall. Coagulation pathways and immune responses contribute to disease development. The role of coagulation factor XII (FXII) in vascular inflammation, however, remains controversial. We here investigated the function of FXII in atherosclerosis using apolipoprotein E and FXII-deficient (F12Apoe) mice. Compared to F12Apoe controls, atherosclerotic lesion formation was reduced in F12Apoe mice. This was associated with a decrease in serum interleukin (IL)-1β and IL-12 levels and reduced expression of pro-inflammatory cytokines in the aorta in atherosclerotic F12Apoe mice, as well as diminished Th1-cell differentiation in the aorta, blood, and lymphoid organs. No changes in circulating bradykinin, thrombin-antithrombin-complexes or plasminogen were observed. Mechanistically, activated FXII (FXIIa) was revealed to directly induce bone marrow-derived macrophages to secrete pro-inflammatory cytokines, including tumour necrosis factor-α, IL-1β, IL-12, and IL-6. Exposure of bone marrow-derived antigen presenting cells to FXIIa similarly induced pro-inflammatory cytokines, and an enhanced capacity to trigger antigen-specific interferon γ-production in CD4 T cells. Notably, bone-marrow derived macrophages were capable of directly activating FXII. Moreover, the induction of cytokine expression by FXIIa in macrophages occurred independently of FXII protease enzymatic activity and was decreased upon phospholipase C treatment, suggesting urokinase-type plasminogen activator receptor (uPAR) to confer FXIIa-induced cell signalling. These data reveal FXII to play an important role in atherosclerotic lesion formation by functioning as a strong inducer of pro-inflammatory cytokines in antigen-presenting cells. Targeting of FXII may thus be a promising approach for treating cardiovascular disease.
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http://dx.doi.org/10.1160/TH16-06-0466DOI Listing
January 2017

Inhibition of cardiac CaMKII to cure heart failure: step by step towards translation?

Basic Res Cardiol 2016 11 28;111(6):66. Epub 2016 Sep 28.

Comprehensive Heart Failure Center, Versbacher Strasse. 9, 97078, Würzburg, Germany.

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http://dx.doi.org/10.1007/s00395-016-0582-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040741PMC
November 2016

Analyzing ERK 1/2 signalling and targets.

Mol Biosyst 2016 07;12(8):2436-46

Department of Bioinformatics, Biocenter, University of Würzburg, Würzburg, Germany.

The ERK cascade (e.g. Raf-1) protects the heart from cell death and ischemic injury but can also turn maladaptive. Furthermore, an additional autophosphorylation of ERK2 at Thr188 (Erk1 at Thr208) allows ERK to phosphorylate nuclear targets involved in hypertrophy, stressing this additional phosphorylation as a promising pharmacological target. An in silico model was assembled and setup to reproduce different phosphorylation states of ERK 1/2 and various types of stimuli (hypertrophic versus non-hypertrophic). Synergistic and antagonistic receptor stimuli can be predicted in a semi-quantitative model, simulated time courses were experimentally validated. Furthermore, we detected new targets of ERK 1/2, which possibly contribute to the development of pathological hypertrophy. In addition we modeled further interaction partners involved in the protective and maladaptive cascade. Experimental validation included different gene expression data sets supporting key components and novel interaction partners as well as time courses in chronic heart failure.
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http://dx.doi.org/10.1039/c6mb00255bDOI Listing
July 2016

β-Adrenoceptor-mediated Relaxation of Urinary Bladder Muscle in β2-Adrenoceptor Knockout Mice.

Front Pharmacol 2016 9;7:118. Epub 2016 May 9.

Department of Physiology, Faculty of Medicine Carl Gustav Carus, Dresden University of Technology Dresden, Germany.

Background And Objective: In order to characterize the β-adrenoceptor (AR) subtypes involved in agonist-stimulated relaxation of murine urinary bladder we studied the effects of (-)-isoprenaline and CL 316,243 on tonic contraction and spontaneous contractions in detrusor strips of wild-type (WT) and β2-AR knockout (β2-AR KO) mice.

Materials And Methods: Urinary bladders were isolated from male WT and β2-AR KO mice. β-AR subtype expression was determined with quantitative real-time PCR. Intact muscle strips pre-contracted with KCl (40 mM) were exposed to cumulatively increasing concentrations of (-)-isoprenaline or β3-AR agonist CL 316,243 in the presence and absence of the subtype-selective β-AR blockers CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs).

Results: Quantitative real-time PCR confirmed lack of β2-AR expression in bladder tissue from β2-AR KO mice. In isolated detrusor strips, pre-contraction with KCl increased basal tone and enhanced spontaneous activity significantly more in β2-AR KO than in WT. (-)-Isoprenaline relaxed tonic tension and attenuated spontaneous activity with similar potency, but the concentrations required were two orders of magnitude higher in β2-AR KO than WT. The concentration-response curves (CRCs) for relaxation were not affected by CGP 20712A (300 nM), but were shifted to the right by ICI 118,551 (50 nM) and L748,337 (10 μM). The -logEC50 values for (-)-isoprenaline in WT and β2-AR KO tissue were 7.98 and 6.00, respectively, suggesting a large receptor reserve of β2-AR. (-)-CL 316,243 relaxed detrusor and attenuated spontaneous contractions from WT and β2-AR KO mice with a potency corresponding to the drug's affinity for β3-AR. L743,337 shifted the CRCs to the right.

Conclusion: Our findings in β2-AR KO mice suggest that there is a large receptor reserve for β2-AR in WT mice so that this β-AR subtype will mediate relaxation of tone and attenuation of spontaneous activity under physiological conditions. Nevertheless, upon removal of this reserve, β3-AR can also mediate murine detrusor relaxation.
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http://dx.doi.org/10.3389/fphar.2016.00118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860462PMC
May 2016

β-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle.

Nature 2016 Mar 23;531(7596):661-4. Epub 2016 Mar 23.

Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany.

(β-)Arrestins are important regulators of G-protein-coupled receptors (GPCRs). They bind to active, phosphorylated GPCRs and thereby shut off 'classical' signalling to G proteins, trigger internalization of GPCRs via interaction with the clathrin machinery and mediate signalling via 'non-classical' pathways. In addition to two visual arrestins that bind to rod and cone photoreceptors (termed arrestin1 and arrestin4), there are only two (non-visual) β-arrestin proteins (β-arrestin1 and β-arrestin2, also termed arrestin2 and arrestin3), which regulate hundreds of different (non-visual) GPCRs. Binding of these proteins to GPCRs usually requires the active form of the receptors plus their phosphorylation by G-protein-coupled receptor kinases (GRKs). The binding of receptors or their carboxy terminus as well as certain truncations induce active conformations of (β-)arrestins that have recently been solved by X-ray crystallography. Here we investigate both the interaction of β-arrestin with GPCRs, and the β-arrestin conformational changes in real time and in living human cells, using a series of fluorescence resonance energy transfer (FRET)-based β-arrestin2 biosensors. We observe receptor-specific patterns of conformational changes in β-arrestin2 that occur rapidly after the receptor-β-arrestin2 interaction. After agonist removal, these changes persist for longer than the direct receptor interaction. Our data indicate a rapid, receptor-type-specific, two-step binding and activation process between GPCRs and β-arrestins. They further indicate that β-arrestins remain active after dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. Thus, GPCRs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signalling.
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http://dx.doi.org/10.1038/nature17198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5157050PMC
March 2016