Publications by authors named "Bernd K Fleischmann"

137 Publications

Proximity to injury, but neither number of nuclei nor ploidy define pathological adaptation and plasticity in cardiomyocytes.

J Mol Cell Cardiol 2021 Mar 5;152:95-104. Epub 2020 Dec 5.

Institute for Cardiovascular Physiology, Goethe University, Frankfurt am Main, Germany; German Center of Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany; Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Germany. Electronic address:

The adult mammalian heart consists of mononuclear and binuclear cardiomyocytes (CMs) with various ploidies. However, it remains unclear whether a variation in ploidy or number of nuclei is associated with distinct functions and injury responses in CMs, including regeneration. Therefore, we investigated transcriptomes and cellular as well as nuclear features of mononucleated and binucleated CMs in adult mouse hearts with and without injury. To be able to identify the role of ploidy we analyzed control and failing human ventricular CMs because human CMs show a larger and disease-sensitive degree of polyploidization. Using transgenic Myh6-H2BmCh to identify mononucleated and binucleated mouse CMs, we found that cellular volume and RNA content were similar in both. On average nuclei of mononuclear CMs showed a 2-fold higher ploidy, as compared to binuclear CMs indicating that most mononuclear CMs are tetraploid. After myocardial infarction mononucleated and binucleated CMs in the border zone of the lesion responded with hypertrophy and corresponding changes in gene expression, as well as a low level of induction of cell cycle gene expression. Human CMs allowed us to study a wide range of polyploidy spanning from 2n to 16n. Notably, basal as well as pathological gene expression signatures and programs in failing CMs proved to be independent of ploidy. In summary, gene expression profiles were induced in proximity to injury, but independent of number of nuclei or ploidy levels in CMs.
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http://dx.doi.org/10.1016/j.yjmcc.2020.11.012DOI Listing
March 2021

Heterotrimeric G Protein Subunit Gαq Is a Master Switch for Gβγ-Mediated Calcium Mobilization by Gi-Coupled GPCRs.

Mol Cell 2020 12 16;80(6):940-954.e6. Epub 2020 Nov 16.

Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany. Electronic address:

Mechanisms that control mobilization of cytosolic calcium [Ca] are key for regulation of numerous eukaryotic cell functions. One such paradigmatic mechanism involves activation of phospholipase Cβ (PLCβ) enzymes by G protein βγ subunits from activated Gα-Gβγ heterotrimers. Here, we report identification of a master switch to enable this control for PLCβ enzymes in living cells. We find that the Gα-Gβγ-PLCβ-Ca signaling module is entirely dependent on the presence of active Gα. If Gα is pharmacologically inhibited or genetically ablated, Gβγ can bind to PLCβ but does not elicit Ca signals. Removal of an auto-inhibitory linker that occludes the active site of the enzyme is required and sufficient to empower "stand-alone control" of PLCβ by Gβγ. This dependence of Gi-Gβγ-Ca on Gα places an entire signaling branch of G-protein-coupled receptors (GPCRs) under hierarchical control of Gq and changes our understanding of how Gi-GPCRs trigger [Ca] via PLCβ enzymes.
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http://dx.doi.org/10.1016/j.molcel.2020.10.027DOI Listing
December 2020

Sensitive LC-MS/MS Method for the Quantification of Macrocyclic Gα Protein Inhibitors in Biological Samples.

Front Chem 2020 24;8:833. Epub 2020 Sep 24.

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, Bonn, Germany.

The cyclic depsipeptide FR900359 (FR) isolated from the plant and produced by endosymbiotic bacteria acts as a selective Gq protein inhibitor. It is a powerful tool to study G protein-coupled receptor signaling, and has potential as a novel drug for the treatment of pulmonary diseases and cancer. For pharmacokinetic studies, sensitive quantitative measurements of drug levels are required. In the present study we established an LC-MS/MS method to detect nanomolar concentrations of FR and the structurally related natural product YM-254890 (YM) in biological samples. HPLC separation coupled to ESI-QTOF-MS and UV-VIS detection was applied. For identification and quantification, the extract ion chromatogram (EIC) of M+1 was evaluated. Limits of detection (LOD) of 0.53-0.55 nM and limits of quantification (LOQ) of 1.6-1.7 nM were achieved for both FR and YM. This protocol was subsequently applied to determine FR concentrations in mouse organs and tissues after peroral application of the drug. A three-step liquid-liquid extraction protocol was established, which resulted in adequate recovery rates of typically around 50%. The results indicated low peroral absorption of FR. Besides the gut, highest concentrations were determined in eye and kidney. The developed analytical method will be useful for preclinical studies to evaluate these potent Gq protein inhibitors, which may have potential as future drugs for complex diseases.
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http://dx.doi.org/10.3389/fchem.2020.00833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540253PMC
September 2020

Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes.

Cell Stem Cell 2020 07;27(1):50-63.e5

Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e., 100- to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3β inhibition suppresses CM maturation, while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10 to 25 times greater expansion beyond GSK-3β inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from yes-associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In summary, we uncovered a molecular interplay that enables massive hiPSC-CM expansion for large-scale drug screening and tissue engineering applications.
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http://dx.doi.org/10.1016/j.stem.2020.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334437PMC
July 2020

Adenosine/A2B Receptor Signaling Ameliorates the Effects of Aging and Counteracts Obesity.

Cell Metab 2020 Jul 25;32(1):56-70.e7. Epub 2020 Jun 25.

Institute of Pharmacology and Toxicology, University Hospital, University of Bonn, 53127 Bonn, Germany. Electronic address:

The combination of aging populations with the obesity pandemic results in an alarming rise in non-communicable diseases. Here, we show that the enigmatic adenosine A2B receptor (A2B) is abundantly expressed in skeletal muscle (SKM) as well as brown adipose tissue (BAT) and might be targeted to counteract age-related muscle atrophy (sarcopenia) as well as obesity. Mice with SKM-specific deletion of A2B exhibited sarcopenia, diminished muscle strength, and reduced energy expenditure (EE), whereas pharmacological A2B activation counteracted these processes. Adipose tissue-specific ablation of A2B exacerbated age-related processes and reduced BAT EE, whereas A2B stimulation ameliorated obesity. In humans, A2B expression correlated with EE in SKM, BAT activity, and abundance of thermogenic adipocytes in white fat. Moreover, A2B agonist treatment increased EE from human adipocytes, myocytes, and muscle explants. Mechanistically, A2B forms heterodimers required for adenosine signaling. Overall, adenosine/A2B signaling links muscle and BAT and has both anti-aging and anti-obesity potential.
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http://dx.doi.org/10.1016/j.cmet.2020.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437516PMC
July 2020

Tetrahydroimidazo[1,2-a]pyrazine Derivatives: Synthesis and Evaluation as Gα -Protein Ligands.

Chemistry 2020 Oct 7;26(55):12615-12623. Epub 2020 Sep 7.

Pharmaceutical Institute, Department of Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany.

The 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine derivative BIM-46174 and its dimeric form BIM-46187 (1) are heterocyclized dipeptides that belong to the very few cell-permeable compounds known to preferentially silence Gα proteins. To explore the chemical space of Gα inhibitors of the BIM chemotype, a combinatorial approach was conducted towards a library of BIM molecules. This library was evaluated in a second messenger-based fluorescence assay to analyze the activity of Gα proteins through the determination of intracellular myo-inositol 1-phosphate. Structure-activity relationships were deduced and structural requirements for biological activity obtained, which were (i) a redox reactive thiol/disulfane substructure, (ii) an N-terminal basic amino group, (iii) a cyclohexylalanine moiety, and (iv) a bicyclic skeleton. Active compounds exhibited cellular toxicity, which was investigated in detail for the prototypical inhibitor 1. This compound affects the structural cytoskeletal dynamics in a Gα -independent manner.
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http://dx.doi.org/10.1002/chem.202001446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590114PMC
October 2020

Cell-permeable high-affinity tracers for G proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors.

Br J Pharmacol 2020 04 11;177(8):1898-1916. Epub 2020 Feb 11.

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany.

Background And Purpose: G proteins are intracellular switches that transduce and amplify extracellular signals from GPCRs. The G protein subtypes, which are coupled to PLC activation, can act as oncogenes, and their expression was reported to be up-regulated in cancer and inflammatory diseases. G inhibition may be an efficient therapeutic strategy constituting a new level of intervention. However, diagnostic tools and therapeutic drugs for G proteins are lacking.

Experimental Approach: We have now developed G -specific, cell-permeable H-labelled high-affinity probes based on the macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM). The tracers served to specifically label and quantify G proteins in their native conformation in cells and tissues with high accuracy.

Key Results: FR and YM displayed low nanomolar affinity for Gα , Gα and Gα expressed in CRISPR/Cas9 Gα -knockout cells, but not for Gα . The two structurally very similar tracers showed strikingly different dissociation kinetics, which is predicted to result in divergent biological effects. Computational studies suggested a "dowel" effect of the pseudoirreversibly binding FR. A high-throughput binding assay led to the discovery of novel G inhibitors, which inhibited G signalling in recombinant cells and primary murine brown adipocytes, resulting in enhanced differentiation.

Conclusions And Implications: The Gq protein inhibitors YM and FR are pharmacologically different despite similar structures. The new versatile tools and powerful assays will contribute to the advancement of the rising field of G protein research.
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http://dx.doi.org/10.1111/bph.14960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070167PMC
April 2020

In vivo detection of programmed cell death during mouse heart development.

Cell Death Differ 2020 04 30;27(4):1398-1414. Epub 2019 Sep 30.

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

Despite the great progress on the cell biology of programmed cell death (PCD), its incidence and exact time course during embryonic and particular heart development are still unclear. This is also due to the lack of models enabling to directly identify and monitor PCD cells at different time points in vivo. Herein we report generation of transgenic murine embryonic stem cell and mouse models expressing secreted Annexin V-YFP under control of the CAG promoter. This enables to visualize and quantify PCD in vitro and in vivo during embryonic development. At early embryonic stages we found Annexin V-YFP fluorescent cells in known areas of PCD, such as the otic ring and at the site of neural tube closing, underscoring its specificity for detection of PCD. We have focused our detailed analysis primarily on PCD in the embryonic heart for a better understanding of its role during development. Our findings reveal that PCD peaks at early stages of cardiogenesis (E9.5-E13.5) and strongly decreases thereafter. Moreover, the PCD cells in the heart are predominantly cardiomyocytes, and an unexpected area of prominent cardiac PCD are the ventricular trabeculae (E9.5-E14.5). Thus, the sA5-YFP mouse line provides novel insight into the incidence and relevance of cardiac PCD during embryonic development ex- and in vivo.
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http://dx.doi.org/10.1038/s41418-019-0426-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205869PMC
April 2020

Optogenetic stimulation of G-signaling in the heart with high spatio-temporal precision.

Nat Commun 2019 03 20;10(1):1281. Epub 2019 Mar 20.

Institute of Physiology I, Medical Faculty, University of Bonn, 53127, Bonn, Germany.

The standard technique for investigating adrenergic effects on heart function is perfusion with pharmaceutical agonists, which does not provide high temporal or spatial precision. Herein we demonstrate that the light sensitive G-protein coupled receptor JellyOp enables optogenetic stimulation of G-signaling in cardiomyocytes and the whole heart. Illumination of transgenic embryonic stem cell-derived cardiomyocytes or of the right atrium of mice expressing JellyOp elevates cAMP levels and instantaneously accelerates spontaneous beating rates similar to pharmacological β-adrenergic stimulation. Light application to the dorsal left atrium instead leads to supraventricular extrabeats, indicating adverse effects of localized G-signaling. In isolated ventricular cardiomyocytes from JellyOp mice, we find increased Ca currents, fractional cell shortening and relaxation rates after illumination enabling the analysis of differential G-signaling with high temporal precision. Thus, JellyOp expression allows localized and time-restricted G stimulation and will provide mechanistic insights into different effects of site-specific, long-lasting and pulsatile G activation.
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http://dx.doi.org/10.1038/s41467-019-09322-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426906PMC
March 2019

Treatment with mononuclear cell populations improves post-infarction cardiac function but does not reduce arrhythmia susceptibility.

PLoS One 2019 14;14(2):e0208301. Epub 2019 Feb 14.

Department of Cardiology, University of Bonn, Bonn, Germany.

Background: Clinical and experimental data give evidence that transplantation of stem and progenitor cells in myocardial infarction could be beneficial, although the underlying mechanism has remained elusive. Ventricular tachyarrhythmia is the most frequent and potentially lethal complication of myocardial infarction, but the impact of mono nuclear cells on the incidence of ventricular arrhythmia is still not clear.

Objective: We aimed to characterize the influence of splenic mononuclear cell populations on ventricular arrhythmia after myocardial infarction.

Methods: We assessed electrical vulnerability in vivo in mice with left ventricular cryoinfarction 14 days after injury and intramyocardial injection of specific subpopulations of mononuclear cells (MNCs) (CD11b-positive cells, Sca-1-positive cells, early endothelial progenitor cells (eEPCs)). As positive control group we used embryonic cardiomyocytes (eCMs). Epicardial mapping was performed for analysing conduction velocities in the border zone. Left ventricular function was quantified by echocardiography and left heart catheterization.

Results: In vivo pacing protocols induced ventricular tachycardia (VT) in 30% of non-infarcted mice. In contrast, monomorphic or polymorphic VT could be evoked in 94% of infarcted and vehicle-injected mice (p<0.01). Only transplantation of eCMs prevented post-infarction VT and improved conduction velocities in the border zone in accordance to increased expression of connexin 43. Cryoinfarction resulted in a broad aggravation of left ventricular function. All transplanted cell types augmented left ventricular function to a similar extent.

Conclusions: Transplantation of different MNC populations after myocardial infarction improves left ventricular function similar to effects of eCMs. Prevention of inducible ventricular arrhythmia is only seen after transplantation of eCMs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0208301PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6375577PMC
October 2019

Label-Free Whole Cell Biosensing for High-Throughput Discovery of Activators and Inhibitors Targeting G Protein-Activated Inwardly Rectifying Potassium Channels.

ACS Omega 2018 Nov 5;3(11):14814-14823. Epub 2018 Nov 5.

Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany.

Dynamic mass redistribution (DMR) and cellular dielectric spectroscopy (CDS) are label-free biosensor technologies that capture real-time integrated cellular responses upon exposure to extra- and intracellular stimuli. They register signaling routes that are accompanied by cell shape changes and/or molecular movement of cells proximal to the biosensor to which they are attached. Here, we report the unexpected observation that robust DMR and CDS signatures are also elicited upon direct stimulation of G protein-activated inwardly rectifying potassium (GIRK) channels, which are involved in the regulation of excitability in the heart and brain. Using ML297, a small-molecule GIRK activator, along with channel blockers and cytoskeletal network inhibitors, we found that GIRK activation exerts its effects on cell shape by a mechanism which depends on actin but not the microtubule network. Because label-free real-time biosensing (i) quantitatively determines concentration dependency of GIRK activators, (ii) accurately assesses the impact of GIRK channel blockers, (iii) is high throughput-compatible, and (iv) visualizes previously unknown cellular consequences downstream of direct GIRK activation, we do not only provide a novel experimental strategy for identification of GIRK ligands but also an entirely new angle to probe GIRK (ligand) biology. We envision that DMR and CDS may add to the repertoire of technologies for systematic exploitation of ion channel function and, in turn, to the identification of novel GIRK ligands in order to treat cardiovascular and neurological disorders.
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http://dx.doi.org/10.1021/acsomega.8b02254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289404PMC
November 2018

PECAM/eGFP transgenic mice for monitoring of angiogenesis in health and disease.

Sci Rep 2018 12 4;8(1):17582. Epub 2018 Dec 4.

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

For the monitoring of vascular growth as well as adaptive or therapeutic (re)vascularization endothelial-specific reporter mouse models are valuable tools. However, currently available mouse models have limitations, because not all endothelial cells express the reporter in all developmental stages. We have generated PECAM/eGFP embryonic stem (ES) cell and mouse lines where the reporter gene labels PECAM endothelial cells and vessels with high specificity. Native eGFP expression and PECAM staining were highly co-localized in vessels of various organs at embryonic stages E9.5, E15.5 and in adult mice. Expression was found in large and small arteries, capillaries and in veins but not in lymphatic vessels. Also in the bone marrow arteries and sinusoidal vessel were labeled, moreover, we could detect eGFP in some CD45 hematopoietic cells. We also demonstrate that this labeling is very useful to monitor sprouting in an aortic ring assay as well as vascular remodeling in a murine injury model of myocardial infarction. Thus, PECAM/eGFP transgenic ES cells and mice greatly facilitate the monitoring and quantification of endothelial cells ex vivo and in vivo during development and injury.
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http://dx.doi.org/10.1038/s41598-018-36039-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279819PMC
December 2018

Midbody Positioning and Distance Between Daughter Nuclei Enable Unequivocal Identification of Cardiomyocyte Cell Division in Mice.

Circ Res 2018 10;123(9):1039-1052

From the Institute of Physiology I, Life and Brain Center, Medical Faculty (M.H., A.B., K.K., N.V., B.K.F.), University of Bonn, Germany.

Rationale: New strategies in the field of cardiac regeneration are directed at identifying proliferation-inducing substances to induce regrowth of myocardium. Current screening assays utilize neonatal cardiomyocytes and markers for cytokinesis, such as Aurora B-kinase. However, detection of cardiomyocyte division is complicated because of cell cycle variants, in particular, binucleation.

Objective: To analyze the process of cardiomyocyte binucleation to identify definitive discriminators for cell cycle variants and authentic cardiomyocyte division.

Methods And Results: Herein, we demonstrate by direct visualization of the contractile ring and midbody in Myh6 (myosin, heavy chain 6)-eGFP (enhanced green fluorescent protein)-anillin transgenic mice that cardiomyocyte binucleation starts by formation of a contractile ring. This is followed by irregular positioning of the midbody and movement of the 2 nuclei into close proximity to each other. In addition, the widespread used marker Aurora B-kinase was found to also label binucleating cardiomyocytes, complicating the interpretation of existing screening assays. Instead, atypical midbody positioning and the distance of daughter nuclei on karyokinesis are bona fide markers for cardiomyocyte binucleation enabling to unequivocally discern such events from cardiomyocyte division in vitro and in vivo.

Conclusions: The 2 criteria provide a new method for identifying cardiomyocyte division and should be considered in future studies investigating cardiomyocyte turnover and regeneration after injury, in particular in the postnatal heart to prevent the assignment of false positive proliferation events.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.312792DOI Listing
October 2018

Pathobiology, pathology and genetics of pulmonary hypertension: Update from the Cologne Consensus Conference 2018.

Int J Cardiol 2018 Dec 20;272S:4-10. Epub 2018 Sep 20.

Excellence Cluster Cardiopulmonary System, University of Giessen Lung Center, German Center for Lung Research (DZL), Member of the German Center for Lung Research (DZL), Justus-Liebig-University, Giessen, Germany.

The European guidelines, which focus on clinical aspects of pulmonary hypertension (PH), provide only minimal information about the pathophysiological concepts of PH. Here, we review this topic in greater detail, focusing on specific aspects in the pathobiology, pathology and genetics, which include mechanisms of vascular inflammation, the role of transcription factors, ion channels/ion channel diseases, hypoxic pulmonary vasoconstriction, genetics/epigenetics, metabolic dysfunction, and the potential future role of histopathology of PH in the modern era of PH therapy. In addition to new insights in the pathobiology of this disease, this working group of the Cologne Consensus Conference also highlights novel concepts and potential new therapeutic targets to further improve the treatment options in PAH.
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http://dx.doi.org/10.1016/j.ijcard.2018.09.070DOI Listing
December 2018

A Novel iRFP-Incorporated in vivo Murine Atherosclerosis Imaging System.

Sci Rep 2018 09 28;8(1):14515. Epub 2018 Sep 28.

Laboratory Animal Resource Center, Faculty of Medicine, University of Tsukuba, 1-1-1, Tennodai, Tsukuba, Ibaraki, 305-8575, Japan.

By using near-infrared fluorescent protein (iRFP)-expressing hematopoietic cells, we established a novel, quantitative, in vivo, noninvasive atherosclerosis imaging system. This murine atherosclerosis imaging approach targets macrophages expressing iRFP in plaques. Low-density lipoprotein receptor-deficient (LDLR) mice transplanted with beta-actin promoter-derived iRFP transgenic (TG) mouse bone marrow (BM) cells (iRFP → LDLR) were used. Atherosclerosis was induced by a nonfluorescent 1.25% cholesterol diet (HCD). Atherosclerosis was compared among the three differently induced mouse groups. iRFP → LDLR mice fed a normal diet (ND) and LDLR mice transplanted with wild-type (WT) BM cells were used as controls. The in vivo imaging system (IVIS) detected an enhanced iRFP signal in the thoracic aorta of HCD-fed iRFP → LDLR mice, whereas iRFP signals were not observed in the control mice. Time-course imaging showed a gradual increase in the signal area, which was correlated with atherosclerotic plaque progression. Oil red O (ORO) staining of aortas and histological analysis of plaques confirmed that the detected signal was strictly emitted from plaque-positive areas of the aorta. Our new murine atherosclerosis imaging system can noninvasively image atherosclerotic plaques in the aorta and generate longitudinal data, validating the ability of the system to monitor lesion progression.
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http://dx.doi.org/10.1038/s41598-018-32456-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162307PMC
September 2018

Sonic hedgehog signaling regulates the mammalian cardiac regenerative response.

J Mol Cell Cardiol 2018 10 17;123:180-184. Epub 2018 Sep 17.

Center for Molecular Medicine, National Heart, Lung and Blood Institute, NIH, Bethesda, MD 20892, USA; Aging Institute of UPMC and the University of Pittsburgh, Pittsburgh, PA 15219, USA. Electronic address:

Certain organisms, including zebrafish, are capable of complete cardiac regeneration in response to injury. This response has also been observed in newborn mice, although in this case, the regenerative capacity is lost at approximately one week of age. The mechanisms regulating this short temporal window of cardiac regeneration in mice are not well understood. Here, we show that sonic hedgehog (Shh) signaling modulates the neonatal mouse regenerative response. In particular, we demonstrate that following apical resection of the heart on postnatal day 1, mice activate Shh ligand expression and downstream signaling. This response is largely absent when surgery is performed on non-regenerative, postnatal day 7 pups. Furthermore, an enhanced cardiac regeneration response was detected in ptch heterozygous mice which have a genetically-based constitutive increase in Shh signaling. We further show that Shh ligand is produced in the myocardium by non-myocytes and appears to regulate cardiomyocyte proliferation, as well as the recruitment of monocytes/macrophages to the regenerating area. Finally, we demonstrate that a small molecule activator of Shh signaling promotes heart regeneration, whereas an inhibitor of Shh signaling impairs the regenerative response. Together, these results implicate Shh signaling as a regulator of mammalian heart regeneration and suggest that modulating this pathway may lead to new potential therapies for cardiovascular diseases.
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http://dx.doi.org/10.1016/j.yjmcc.2018.09.005DOI Listing
October 2018

The Transcription Factor ETV1 Induces Atrial Remodeling and Arrhythmia.

Circ Res 2018 08;123(5):550-563

From the Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine (C.R., S.R., A.L., M.B., M.S., R.G., T.B., T.S., S.M., L.H.).

Rationale: Structural and electrophysiological remodeling of the atria are recognized consequences of sustained atrial arrhythmias, such as atrial fibrillation. The identification of underlying key molecules and signaling pathways has been challenging because of the changing cell type composition during structural remodeling of the atria.

Objective: Thus, the aims of our study were (1) to search for transcription factors and downstream target genes, which are involved in atrial structural remodeling, (2) to characterize the significance of the transcription factor ETV1 (E twenty-six variant 1) in atrial remodeling and arrhythmia, and (3) to identify ETV1-dependent gene regulatory networks in atrial cardiac myocytes.

Methods And Results: The transcription factor ETV1 was significantly upregulated in atrial tissue from patients with permanent atrial fibrillation. Mice with cardiac myocyte-specific overexpression of ETV1 under control of the myosin heavy chain promoter developed atrial dilatation, fibrosis, thrombosis, and arrhythmia. Cardiac myocyte-specific ablation of ETV1 in mice did not alter cardiac structure and function at baseline. Treatment with Ang II (angiotensin II) for 2 weeks elicited atrial remodeling and fibrosis in control, but not in ETV1-deficient mice. To identify ETV1-regulated genes, cardiac myocytes were isolated and purified from mouse atrial tissue. Active cis-regulatory elements in mouse atrial cardiac myocytes were identified by chromatin accessibility (assay for transposase-accessible chromatin sequencing) and the active chromatin modification H3K27ac (chromatin immunoprecipitation sequencing). One hundred seventy-eight genes regulated by Ang II in an ETV1-dependent manner were associated with active cis-regulatory elements containing ETV1-binding sites. Various genes involved in Ca handling or gap junction formation ( Ryr2, Jph2, Gja5), potassium channels ( Kcnh2, Kcnk3), and genes implicated in atrial fibrillation ( Tbx5) were part of this ETV1-driven gene regulatory network. The atrial ETV1-dependent transcriptome in mice showed a significant overlap with the human atrial proteome of patients with permanent atrial fibrillation.

Conclusions: This study identifies ETV1 as an important component in the pathophysiology of atrial remodeling associated with atrial arrhythmias.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.313036DOI Listing
August 2018

Overexpression of Cx43 in cells of the myocardial scar: Correction of post-infarct arrhythmias through heterotypic cell-cell coupling.

Sci Rep 2018 05 8;8(1):7145. Epub 2018 May 8.

Institute of Physiology I, Life&Brain Center, Medical Faculty, University of Bonn, Sigmund Freud Str. 25, 53127, Bonn, Germany.

Ventricular tachycardia (VT) is the most common and potentially lethal complication following myocardial infarction (MI). Biological correction of the conduction inhomogeneity that underlies re-entry could be a major advance in infarction therapy. As minimal increases in conduction of infarcted tissue markedly influence VT susceptibility, we reasoned that enhanced propagation of the electrical signal between non-excitable cells within a resolving infarct might comprise a simple means to decrease post-infarction arrhythmia risk. We therefore tested lentivirus-mediated delivery of the gap-junction protein Connexin 43 (Cx43) into acute myocardial lesions. Cx43 was expressed in (myo)fibroblasts and CD45 cells within the scar and provided prominent and long lasting arrhythmia protection in vivo. Optical mapping of Cx43 injected hearts revealed enhanced conduction velocity within the scar, indicating Cx43-mediated electrical coupling between myocytes and (myo)fibroblasts. Thus, Cx43 gene therapy, by direct in vivo transduction of non-cardiomyocytes, comprises a simple and clinically applicable biological therapy that markedly reduces post-infarction VT.
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http://dx.doi.org/10.1038/s41598-018-25147-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940892PMC
May 2018

Intra-Cardiac Release of Extracellular Vesicles Shapes Inflammation Following Myocardial Infarction.

Circ Res 2018 06 28;123(1):100-106. Epub 2018 Mar 28.

From the INSERM UMR-S 970, Paris Cardiovascular Research Center, France (X.L., I.Z., C.D., M.Y., K.-Y.H., P.K., M.K., J.V., P.M., J.-S.S., C.M.B.)

Rationale: A rapid and massive influx of inflammatory cells occurs into ischemic area after myocardial infarction (MI), resulting in local release of cytokines and growth factors. Yet, the mechanisms regulating their production are not fully explored. The release of extracellular vesicles (EVs) in the interstitial space curbs important biological functions, including inflammation, and influences the development of cardiovascular diseases. To date, there is no evidence for in situ release of cardiac EVs after MI.

Objective: The present study tested the hypothesis that local EV generation in the infarcted heart coordinates cardiac inflammation after MI.

Methods And Results: Coronary artery ligation in mice transiently increases EV levels in the left ventricle when compared with sham animals. EVs from infarcted hearts were characterized as large vesicles (252±18 nm) expressing cardiomyocyte and endothelial markers and small EVs (118±4 nm) harboring exosomal markers, such as CD (cluster of differentiation) 63 and CD9. Cardiac large EVs generated after MI, but not small EVs or sham EVs, increased the release of IL (interleukin)-6, CCL (chemokine ligand) 2, and CCL7 from fluorescence-activated cell-sorted Ly6C cardiac monocytes. EVs of similar diameter were also isolated from fragments of interventricular septum obtained from patients undergoing aortic valve replacement, thus supporting the clinical relevance of our findings in mice.

Conclusions: The present study demonstrates that acute MI transiently increases the generation of cardiac EVs characterized as both exosomes and microvesicles, originating mainly from cardiomyocytes and endothelial cells. EVs accumulating in the ischemic myocardium are rapidly taken up by infiltrating monocytes and regulate local inflammatory responses.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.311326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023578PMC
June 2018

Reactivation of the Nkx2.5 cardiac enhancer after myocardial infarction does not presage myogenesis.

Cardiovasc Res 2018 07;114(8):1098-1114

Department of Cardiovascular Surgery, German Heart Center Munich at the Technische Universität München, Lazarettstraße 36, 80636 Munich, Germany.

Aims: The contribution of resident stem or progenitor cells to cardiomyocyte renewal after injury in adult mammalian hearts remains a matter of considerable debate. We evaluated a cell population in the adult mouse heart induced by myocardial infarction (MI) and characterized by an activated Nkx2.5 enhancer element that is specific for multipotent cardiac progenitor cells (CPCs) during embryonic development. We hypothesized that these MI-induced cells (MICs) harbour cardiomyogenic properties similar to their embryonic counterparts.

Methods And Results: MICs reside in the heart and mainly localize to the infarction area and border zone. Interestingly, gene expression profiling of purified MICs 1 week after infarction revealed increased expression of stem cell markers and embryonic cardiac transcription factors (TFs) in these cells as compared to the non-mycoyte cell fraction of adult hearts. A subsequent global transcriptome comparison with embryonic CPCs and fibroblasts and in vitro culture of MICs unveiled that (myo-)fibroblastic features predominated and that cardiac TFs were only expressed at background levels.

Conclusions: Adult injury-induced reactivation of a cardiac-specific Nkx2.5 enhancer element known to specifically mark myocardial progenitor cells during embryonic development does not reflect hypothesized embryonic cardiomyogenic properties. Our data suggest a decreasing plasticity of cardiac progenitor (-like) cell populations with increasing age. A re-expression of embryonic, stem or progenitor cell features in the adult heart must be interpreted very carefully with respect to the definition of cardiac resident progenitor cells. Albeit, the abundance of scar formation after cardiac injury suggests a potential to target predestinated activated profibrotic cells to push them towards cardiomyogenic differentiation to improve regeneration.
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http://dx.doi.org/10.1093/cvr/cvy069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279078PMC
July 2018

In Vivo Labeling by CD73 Marks Multipotent Stromal Cells and Highlights Endothelial Heterogeneity in the Bone Marrow Niche.

Cell Stem Cell 2018 02;22(2):262-276.e7

Institute of Physiology I, Life&Brain Center, Medical Faculty, University of Bonn, 53105 Bonn, Germany. Electronic address:

Despite much work studying ex vivo multipotent stromal cells (MSCs), the identity and characteristics of MSCs in vivo are not well defined. Here, we generated a CD73-EGFP reporter mouse to address these questions and found EGFP MSCs in various organs. In vivo, EGFP mesenchymal cells were observed in fetal and adult bones at proliferative ossification sites, while in solid organs EGFP cells exhibited a perivascular distribution pattern. EGFP cells from the bone compartment could be clonally expanded ex vivo from single cells and displayed trilineage differentiation potential. Moreover, in the central bone marrow CD73-EGFP specifically labeled sinusoidal endothelial cells, thought to be a critical component of the hematopoietic stem cell niche. Purification and molecular characterization of this CD73-EGFP population revealed an endothelial subtype that also displays a mesenchymal signature, highlighting endothelial cell heterogeneity in the marrow. Thus, the CD73-EGFP mouse is a powerful tool for studying MSCs and sinusoidal endothelium.
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http://dx.doi.org/10.1016/j.stem.2018.01.008DOI Listing
February 2018

Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system.

Angiogenesis 2018 05 7;21(2):349-361. Epub 2018 Feb 7.

Institute of Physiology I, Life and Brain Center, Medical Faculty, University of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany.

Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP signals specifically in Ki67/PECAM endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.
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http://dx.doi.org/10.1007/s10456-018-9601-1DOI Listing
May 2018

Improved heart repair upon myocardial infarction: Combination of magnetic nanoparticles and tailored magnets strongly increases engraftment of myocytes.

Biomaterials 2018 Feb 15;155:176-190. Epub 2017 Nov 15.

Department of Cardiac Surgery, Medical Faculty, University of Bonn, Sigmund Freud Str. 25, 53105 Bonn, Germany. Electronic address:

Cell replacement in the heart is considered a promising strategy for the treatment of post-infarct heart failure. Direct intramyocardial injection of cells proved to be the most effective application route, however, engraftment rates are very low (<5%) strongly hampering its efficacy. Herein we combine magnetic nanoparticle (MNP) loading of EGFP labeled embryonic cardiomyocytes (eCM) and embryonic stem cell-derived cardiomyocytes (ES-CM) with application of custom designed magnets to enhance their short and long-term engraftment. To optimize cellular MNP uptake and magnetic force within the infarct area, first numerical simulations and experiments were performed in vitro. All tested cell types could be loaded efficiently with SOMag5-MNP (200 pg/cell) without toxic side effects. Application of a 1.3 T magnet at 5 mm distance from the heart for 10 min enhanced engraftment of both eCM and ES-CM by approximately 7 fold at 2 weeks and 3.4 fold (eCM) at 8 weeks after treatment respectively and also strongly improved left ventricular function at all time points. As underlying mechanisms we found that application of the magnetic field prevented the initial dramatic loss of cells via the injection channel. In addition, grafted eCM displayed higher proliferation and lower apoptosis rates. Electron microscopy revealed better differentiation of engrafted eCM, formation of cell to cell contacts and more physiological matrix formation in magnet-treated grafts. These results were corroborated by gene expression data. Thus, combination of MNP-loaded cells and magnet-application strongly increases long-term engraftment of cells addressing a major shortcoming of cardiomyoplasty.
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http://dx.doi.org/10.1016/j.biomaterials.2017.11.012DOI Listing
February 2018

PDK4 Inhibits Cardiac Pyruvate Oxidation in Late Pregnancy.

Circ Res 2017 Dec 19;121(12):1370-1378. Epub 2017 Sep 19.

From the Cardiovascular Institute, Beth Israel Deaconess Medical Center, Boston, MA (L.X.L., F.D.); Corrigan Minehan Heart Center, Massachusetts General Hospital, Boston (L.X.L., F.D., M.C.C., S.D., A.R.); Division of Cardiovascular Disease, University of Alabama at Birmingham (G.C.R.); Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia (S.Y., J.L., S.W., M.M., Z.A.); Lewis-Sigler Institute for Integrative Genomics, Princeton University, NJ (W.L., C.J., J.D.R.); and Institute of Physiology I, Life and Brain Center, Medical Faculty, University of Bonn, Germany (M.H., B.K.F.).

Rationale: Pregnancy profoundly alters maternal physiology. The heart hypertrophies during pregnancy, but its metabolic adaptations, are not well understood.

Objective: To determine the mechanisms underlying cardiac substrate use during pregnancy.

Methods And Results: We use here C glucose, C lactate, and C fatty acid tracing analyses to show that hearts in late pregnant mice increase fatty acid uptake and oxidation into the tricarboxylic acid cycle, while reducing glucose and lactate oxidation. Mitochondrial quantity, morphology, and function do not seem altered. Insulin signaling seems intact, and the abundance and localization of the major fatty acid and glucose transporters, CD36 (cluster of differentiation 36) and GLUT4 (glucose transporter type 4), are also unchanged. Rather, we find that the pregnancy hormone progesterone induces PDK4 (pyruvate dehydrogenase kinase 4) in cardiomyocytes and that elevated PDK4 levels in late pregnancy lead to inhibition of PDH (pyruvate dehydrogenase) and pyruvate flux into the tricarboxylic acid cycle. Blocking PDK4 reverses the metabolic changes seen in hearts in late pregnancy.

Conclusions: Taken together, these data indicate that the hormonal environment of late pregnancy promotes metabolic remodeling in the heart at the level of PDH, rather than at the level of insulin signaling.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.311456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5722682PMC
December 2017

Targeted inhibition of G signaling induces airway relaxation in mouse models of asthma.

Sci Transl Med 2017 Sep;9(407)

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

Obstructive lung diseases are common causes of disability and death worldwide. A hallmark feature is aberrant activation of G protein-dependent signaling cascades. Currently, drugs targeting single G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) are used to reduce airway tone. However, therapeutic efficacy is often limited, because various GPCRs contribute to bronchoconstriction, and chronic exposure to receptor-activating medications results in desensitization. We therefore hypothesized that pharmacological G inhibition could serve as a central mechanism to achieve efficient therapeutic bronchorelaxation. We found that the compound FR900359 (FR), a membrane-permeable inhibitor of G, was effective in silencing G signaling in murine and human airway smooth muscle cells. Moreover, FR both prevented bronchoconstrictor responses and triggered sustained airway relaxation in mouse, pig, and human airway tissue ex vivo. Inhalation of FR in healthy wild-type mice resulted in high local concentrations of the compound in the lungs and prevented airway constriction without acute effects on blood pressure and heart rate. FR administration also protected against airway hyperreactivity in murine models of allergen sensitization using ovalbumin and house dust mite as allergens. Our findings establish FR as a selective G inhibitor when applied locally to the airways of mice in vivo and suggest that pharmacological blockade of G proteins may be a useful therapeutic strategy to achieve bronchorelaxation in asthmatic lung disease.
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http://dx.doi.org/10.1126/scitranslmed.aag2288DOI Listing
September 2017

Heart regeneration and the cardiomyocyte cell cycle.

Pflugers Arch 2018 02 28;470(2):241-248. Epub 2017 Aug 28.

Institute of Physiology I, Life & Brain Center, University of Bonn, Sigmund-Freud-Strasse 25, 53105, Bonn, Germany.

Cardiovascular disease and in particular, heart failure are still main causes of death; therefore, novel therapeutic approaches are urgently needed. Loss of contractile substrate in the heart and limited regenerative capacity of cardiomyocytes are mainly responsible for the poor cardiovascular outcome. This is related to the postmitotic state of differentiated cardiomyocytes, which is partly due to their polyploid nature caused by cell cycle variants. As such, the cardiomyocyte cell cycle is a key player, and its manipulation could be a promising strategy for enhancing the plasticity of the heart by inducing cardiomyocyte proliferation. This review focuses on the cardiac cell cycle and its variants during postnatal growth, the different regenerative responses of the heart in dependance of the developmental stage and on manipulations of the cell cycle. Because a therapeutic goal is to induce authentic cell division in cardiomyocytes, recent experimental approaches following this strategy are also discussed.
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http://dx.doi.org/10.1007/s00424-017-2061-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780532PMC
February 2018

Cardiomyocyte Regeneration: A Consensus Statement.

Circulation 2017 08 6;136(7):680-686. Epub 2017 Jul 6.

From Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg Eppendorf, Hamburg, Germany (T.E.); DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany (T.E.) and partner site Rhein/Main, Bad Nauheim, Germany (T.B.); Institute of Molecular Cardiology, University of Louisville, Louisville, KY (R.B.); Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany (T.B.); Department of Internal Medicine II, University of Giessen, Germany (T.B.); German Center for Lung Research (DZHL), Giessen/Marburg Bad Nauheim, Bad Nauheim, Germany (T.B.); Krannert Institute of Cardiology and Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis (L.J.F.); Institute of Physiology I, Life and Brain Center, Medical Faculty, University of Bonn, Germany (B.K.F.); Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden (J.F.); International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy (M.G.); Donald Soffer Endowed Program in Regenerative Medicine, Miller School of Medicine, Miami, FL (J.M.H.); Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD (J.M.H.); Department of Physiology, Lewis Katz School of Medicine, Temple University, Philadelphia, PA (S.H.); Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA (R.T.L.); Cedars-Sinai Heart Institute, Los Angeles, CA (E.M.); Cardiomyocyte Renewal Laboratory, Texas Heart Institute, Houston (J.F.M.); Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX (J.F.M.); Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati, OH (J.D.M.); Departments of Pathology, Bioengineering, and Medicine/Cardiology, Institute for Stem Cell and Regenerative Medicine, and Center for Cardiovascular Biology, University of Washington, Seattle (C.E.M.); University of Oxford, Department of Physiology, Anatomy and Genetics, United Kingdom (P.R.R.) Regencor, Inc, Los Altos, CA (P.R.-L.); Departments of Internal Medicine (Division of Cardiology) and Molecular Biology, UT Southwestern Medical Center, Dallas, TX (H.A.S., J.A.H.); and Heart Institute, Integrated Regenerative Research Institute, and Biology Department, San Diego State University, CA (M.S.A.).

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http://dx.doi.org/10.1161/CIRCULATIONAHA.117.029343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557671PMC
August 2017

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes.

J Vis Exp 2017 02 24(120). Epub 2017 Feb 24.

Institute of Physiology I, University of Bonn;

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.
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http://dx.doi.org/10.3791/55204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409286PMC
February 2017

Intercellular transfer of miR-126-3p by endothelial microparticles reduces vascular smooth muscle cell proliferation and limits neointima formation by inhibiting LRP6.

J Mol Cell Cardiol 2017 03 28;104:43-52. Epub 2017 Jan 28.

Department of Internal Medicine II, Rheinische Friedrich-Wilhelms University, Bonn, Germany. Electronic address:

Background: Vascular smooth muscle cell (VSMC) proliferation is of importance in the pathogenesis of vascular diseases such as restenosis or atherosclerosis. Endothelial microparticles (EMPs) regulate function and phenotype of target endothelial cells (ECs), but their influence on VSMC biology is unknown. We aim to investigate the role of EMPs in the regulation of vascular smooth muscle cell (VSMC) proliferation and vascular remodeling.

Methods And Results: Systemic treatment of mice with EMPs after vascular injury reduced neointima formation in vivo. In vitro, EMP uptake in VSMCs diminished VSMC proliferation and migration, both pivotal steps in neointima formation. To explore the underlying mechanisms, Taqman microRNA-array was performed and miR-126-3p was identified as the predominantly expressed miR in EMPs. Confocal microscopy revealed an EMP-mediated miR-126 transfer into recipient VSMCs. Expression of miR-126 target protein LRP6, regulating VSMC proliferation, was reduced in VSMCs after EMP treatment. Importantly, genetic regulation of miR-126 in EMPs showed a miR-126-dependent inhibition of LRP6 expression, VSMC proliferation and neointima formation in vitro and in vivo, suggesting a crucial role of miR-126 in EMP-mediated neointima formation reduction. Finally, analysis of miR-126 expression in circulating MPs in 176 patients with coronary artery disease revealed a reduced PCI rate in patients with high miR-126 expression level, supporting a central role for MP-incorporated miR-126 in vascular remodelling.

Conclusion: EMPs reduce VSMC proliferation, migration and subsequent neointima formation by delivering functional miR-126 into recipient VSMCs.
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http://dx.doi.org/10.1016/j.yjmcc.2016.12.005DOI Listing
March 2017

Engineered Context-Sensitive Agonism: Tissue-Selective Drug Signaling through a G Protein-Coupled Receptor.

J Pharmacol Exp Ther 2017 Feb 15;360(2):289-299. Epub 2016 Nov 15.

Pharmacology and Toxicology Section, Institute of Pharmacy, University of Bonn, Bonn, Germany (W.K.S., R.S., J.E., T.B., A.B., K.M.); Institute of Physiology I, Life&Brain Center, Medical Faculty, University of Bonn, Bonn, Germany (D.W., P.S., B.K.F.); Institute of Pharmacology & Toxicology, University of Bonn, Bonn, Germany (M.W., E.S., K.R.); Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Würzburg, Würzburg, Germany (J.K., U.H.); Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica 'Pietro Pratesi,' Università degli Studi di Milano, Milano, Italy (M.D.); Molecular, Cellular, and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn, Bonn, Germany (E.K.); Institute of Physiology II, University of Bonn, Bonn, Germany (R.M.); Center of Pharmacology, University of Cologne, Cologne, Germany (W.K.S.).

Drug discovery strives for selective ligands to achieve targeted modulation of tissue function. Here we introduce engineered context-sensitive agonism as a postreceptor mechanism for tissue-selective drug action through a G protein-coupled receptor. Acetylcholine M-receptor activation is known to mediate, among other actions, potentially dangerous slowing of the heart rate. This unwanted side effect is one of the main reasons that limit clinical application of muscarinic agonists. Herein we show that dualsteric (orthosteric/allosteric) agonists induce less cardiac depression ex vivo and in vivo than conventional full agonists. Exploration of the underlying mechanism in living cells employing cellular dynamic mass redistribution identified context-sensitive agonism of these dualsteric agonists. They translate elevation of intracellular cAMP into a switch from full to partial agonism. Designed context-sensitive agonism opens an avenue toward postreceptor pharmacologic selectivity, which even works in target tissues operated by the same subtype of pharmacologic receptor.
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http://dx.doi.org/10.1124/jpet.116.237149DOI Listing
February 2017