Publications by authors named "Anke Zieseniss"

28 Publications

  • Page 1 of 1

Precisely Tuned Inhibition of HIF Prolyl Hydroxylases Is Key for Cardioprotection After Ischemia.

Circ Res 2021 Apr 25;128(8):1208-1210. Epub 2021 Feb 25.

Institute of Cardiovascular Physiology, University Medical Center Göttingen, Göttingen, Germany (A.J., A.Z., K.B.-C., A.M.V., D.M.K.).

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http://dx.doi.org/10.1161/CIRCRESAHA.120.318216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8048377PMC
April 2021

Epigenetic gene expression links heart failure to memory impairment.

EMBO Mol Med 2021 Mar 20;13(3):e11900. Epub 2021 Jan 20.

Department for Systems Medicine and Epigenetics, German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany.

In current clinical practice, care of diseased patients is often restricted to separated disciplines. However, such an organ-centered approach is not always suitable. For example, cognitive dysfunction is a severe burden in heart failure patients. Moreover, these patients have an increased risk for age-associated dementias. The underlying molecular mechanisms are presently unknown, and thus, corresponding therapeutic strategies to improve cognition in heart failure patients are missing. Using mice as model organisms, we show that heart failure leads to specific changes in hippocampal gene expression, a brain region intimately linked to cognition. These changes reflect increased cellular stress pathways which eventually lead to loss of neuronal euchromatin and reduced expression of a hippocampal gene cluster essential for cognition. Consequently, mice suffering from heart failure exhibit impaired memory function. These pathological changes are ameliorated via the administration of a drug that promotes neuronal euchromatin formation. Our study provides first insight to the molecular processes by which heart failure contributes to neuronal dysfunction and point to novel therapeutic avenues to treat cognitive defects in heart failure patients.
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http://dx.doi.org/10.15252/emmm.201911900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933944PMC
March 2021

O affects mitochondrial functionality ex vivo.

Redox Biol 2019 04 23;22:101152. Epub 2019 Feb 23.

Institute for Cardiovascular Physiology, University Medical Center Göttingen, Georg-August-University, Humbdoltallee 23, 37077 Göttingen, Germany. Electronic address:

Mitochondria have originated in eukaryotic cells by endosymbiosis of a specialized prokaryote approximately 2 billion years ago. They are essential for normal cell function by providing energy through their role in oxidizing carbon substrates. Glutathione (GSH) is a major thiol-disulfide redox buffer of the cell including the mitochondrial matrix and intermembrane space. We have generated cardiomyocyte-specific Grx1-roGFP2 GSH redox potential (E) biosensor mice in the past, in which the sensor is targeted to the mitochondrial matrix. Using this mouse model a distinct E of the mitochondrial matrix (-278.9 ± 0.4 mV) in isolated cardiomyocytes is observed. When analyzing the E in isolated mitochondria from the transgenic hearts, however, the E in the mitochondrial matrix is significantly oxidized (-247.7 ± 8.7 mV). This is prevented by adding N-Ethylmaleimide during the mitochondria isolation procedure, which precludes disulfide bond formation. A similar reducing effect is observed by isolating mitochondria in hypoxic (0.1-3% O) conditions that mimics mitochondrial pO levels in cellulo. The reduced E is accompanied by lower ROS production, reduced complex III activity but increased ATP levels produced at baseline and after stimulation with succinate/ADP. Altogether, we demonstrate that oxygenation is an essential factor that needs to be considered when analyzing mitochondrial function ex vivo.
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http://dx.doi.org/10.1016/j.redox.2019.101152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396017PMC
April 2019

Hypoxia suppresses myofibroblast differentiation by changing RhoA activity.

J Cell Sci 2019 02 18;132(5). Epub 2019 Feb 18.

Institute of Cardiovascular Physiology, University Medical Center, Georg-August University Göttingen, 37073 Göttingen, Germany

Fibroblasts show a high range of phenotypic plasticity, including transdifferentiation into myofibroblasts. Myofibroblasts are responsible for generation of the contraction forces that are important for wound healing and scar formation. Overactive myofibroblasts, by contrast, are involved in abnormal scarring. Cell stretching and extracellular signals such as transforming growth factor β can induce the myofibroblastic program, whereas microenvironmental conditions such as reduced tissue oxygenation have an inhibitory effect. We investigated the effects of hypoxia on myofibroblastic properties and linked this to RhoA activity. Hypoxia reversed the myofibroblastic phenotype of primary fibroblasts. This was accompanied by decreased αSMA (ACTA2) expression, alterations in cell contractility, actin reorganization and RhoA activity. We identified a hypoxia-inducible induction of ARHGAP29, which is critically involved in myocardin-related transcription factor-A (MRTF-A) signaling, the differentiation state of myofibroblasts and modulates RhoA activity. This novel link between hypoxia and MRTF-A signaling is likely to be important for ischemia-induced tissue remodeling and the fibrotic response.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.223230DOI Listing
February 2019

Defective Mitochondrial Cardiolipin Remodeling Dampens HIF-1α Expression in Hypoxia.

Cell Rep 2018 10;25(3):561-570.e6

Department of Cellular Biochemistry, University Medical Center Göttingen, GZMB, 37073 Göttingen, Germany.

Mitochondria fulfill vital metabolic functions and act as crucial cellular signaling hubs, integrating their metabolic status into the cellular context. Here, we show that defective cardiolipin remodeling, upon loss of the cardiolipin acyl transferase tafazzin, decreases HIF-1α signaling in hypoxia. Tafazzin deficiency does not affect posttranslational HIF-1α regulation but rather HIF-1α gene expression, a dysfunction recapitulated in iPSC-derived cardiomyocytes from Barth syndrome patients with tafazzin deficiency. RNA-seq analyses confirmed drastically altered signaling in tafazzin mutant cells. In hypoxia, tafazzin-deficient cells display reduced production of reactive oxygen species (ROS) perturbing NF-κB activation and concomitantly HIF-1α gene expression. Tafazzin-deficient mice hearts display reduced HIF-1α levels and undergo maladaptive hypertrophy with heart failure in response to pressure overload challenge. We conclude that defective mitochondrial cardiolipin remodeling dampens HIF-1α signaling due to a lack of NF-κB activation through reduced mitochondrial ROS production, decreasing HIF-1α transcription.
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http://dx.doi.org/10.1016/j.celrep.2018.09.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205837PMC
October 2018

Transgenic Organisms Meet Redox Bioimaging: One Step Closer to Physiology.

Antioxid Redox Signal 2018 08 16;29(6):603-612. Epub 2018 Feb 16.

2 Institute of Cardiovascular Physiology, University Medical Center Göttingen, Georg-August University of Göttingen , Göttingen, Germany .

Significance: Redox signaling is a common mechanism in the cellular response toward a variety of stimuli. For analyzing redox-dependent specific alterations in a cell, genetically encoded biosensors were highly instrumental in the past. To advance the knowledge about the importance of this signaling mechanism in vivo, models that are as close as possible to physiology are needed. Recent Advances: The development of transgenic (tg) redox biosensor animal models has enhanced the knowledge of redox signaling under patho(physio)logical conditions. So far, commonly used small animal models, that is, Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio, and genetically modified mice were employed for redox biosensor transgenesis. However, especially the available mouse models are still limited.

Critical Issues: The analysis of redox biosensor responses in vivo at the tissue level, especially for internal organs, is hampered by the detection limit of the available redox biosensors and microscopy techniques. Recent technical developments such as redox histology and the analysis of cell-type-specific biosensor responses need to be further refined and followed up in a systematic manner.

Future Directions: The usage of tg animal models in the field of redox signaling has helped to answer open questions. Application of the already established models and consequent development of more defined tg models will enable this research area to define the role of redox signaling in (patho)physiology in further depth. Antioxid. Redox Signal. 29, 603-612.
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http://dx.doi.org/10.1089/ars.2017.7469DOI Listing
August 2018

Loss of PHD3 in myeloid cells dampens the inflammatory response and fibrosis after hind-limb ischemia.

Cell Death Dis 2017 08 10;8(8):e2976. Epub 2017 Aug 10.

Institute of Cardiovascular Physiology, University Medical Center, Göttingen, Germany.

Macrophages are essential for the inflammatory response after an ischemic insult and thereby influence tissue recovery. For the oxygen sensing prolyl-4-hydroxylase domain enzyme (PHD) 2 a clear impact on the macrophage-mediated arteriogenic response after hind-limb ischemia has been demonstrated previously, which involves fine tuning a M2-like macrophage population. To analyze the role of PHD3 in macrophages, we performed hind-limb ischemia (ligation and excision of the femoral artery) in myeloid-specific PHD3 knockout mice (PHD3) and analyzed the inflammatory cell invasion, reperfusion recovery and fibrosis in the ischemic muscle post-surgery. In contrast to PHD2, reperfusion recovery and angiogenesis was unaltered in PHD3 compared to WT mice. Macrophages from PHD3 mice showed, however, a dampened inflammatory reaction in the affected skeletal muscle tissues compared to WT controls. This was associated with a decrease in fibrosis and an anti-inflammatory phenotype of the PHD3 macrophages, as well as decreased expression of Cyp2s1 and increased PGE2-secretion, which could be mimicked by PHD3 bone marrow-derived macrophages in serum starvation.
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http://dx.doi.org/10.1038/cddis.2017.375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596563PMC
August 2017

Oxygen-dependent regulation of aquaporin-3 expression.

Hypoxia (Auckl) 2016 21;4:91-97. Epub 2016 Apr 21.

Institute of Cardiovascular Physiology, University Medical Center Göttingen, University of Göttingen, Göttingen, Germany.

The purpose of this study was to investigate whether aquaporin-3 (AQP3) expression is altered in hypoxia and whether hypoxia-inducible transcription factor (HIF)-1 regulates the hypoxic expression. AQP3 mRNA expression was studied in L929 fibrosarcoma cells and in several tissues derived from mice that were subjected to hypoxia. Computational analysis of the AQP3 promoter revealed conserved HIF binding sites within close proximity to the translational start site, and chromatin immunoprecipitation assays confirmed binding of HIF-1α to the endogenous hypoxia response elements. Furthermore, hypoxia resulted in increased expression of AQP3 mRNA in L929 fibrosarcoma cells. Consistently, shRNA-mediated knockdown of HIF-1α greatly reduced the hypoxic induction of AQP3. In addition, mRNA analysis of organs from mice exposed to inspiratory hypoxia demonstrated pronounced hypoxia-inducible expression of AQP3 in the kidney. Overall, our findings suggest that AQP3 expression can be regulated at the transcriptional level and that AQP3 represents a novel HIF-1 target gene.
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http://dx.doi.org/10.2147/HP.S97681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085303PMC
April 2016

PHD2 Is a Regulator for Glycolytic Reprogramming in Macrophages.

Mol Cell Biol 2017 01 19;37(1). Epub 2016 Dec 19.

Institute for Cardiovascular Physiology, Georg August University Göttingen, Göttingen, Germany

The prolyl-4-hydroxylase domain (PHD) enzymes are regarded as the molecular oxygen sensors. There is an interplay between oxygen availability and cellular metabolism, which in turn has significant effects on the functionality of innate immune cells, such as macrophages. However, if and how PHD enzymes affect macrophage metabolism are enigmatic. We hypothesized that macrophage metabolism and function can be controlled via manipulation of PHD2. We characterized the metabolic phenotypes of PHD2-deficient RAW cells and primary PHD2 knockout bone marrow-derived macrophages (BMDM). Both showed typical features of anaerobic glycolysis, which were paralleled by increased pyruvate dehydrogenase kinase 1 (PDK1) protein levels and a decreased pyruvate dehydrogenase enzyme activity. Metabolic alterations were associated with an impaired cellular functionality. Inhibition of PDK1 or knockout of hypoxia-inducible factor 1α (HIF-1α) reversed the metabolic phenotype and impaired the functionality of the PHD2-deficient RAW cells and BMDM. Taking these results together, we identified a critical role of PHD2 for a reversible glycolytic reprogramming in macrophages with a direct impact on their function. We suggest that PHD2 serves as an adjustable switch to control macrophage behavior.
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http://dx.doi.org/10.1128/MCB.00236-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192080PMC
January 2017

Redox Imaging Using Cardiac Myocyte-Specific Transgenic Biosensor Mice.

Circ Res 2016 Oct 23;119(9):1004-1016. Epub 2016 Aug 23.

From the Institute of Cardiovascular Physiology, Georg August University Göttingen, Germany (L.S., A.K., A.Z., A.G., A.J., A.B., M.S.N., D.M.K.); Institute of Pharmacology, Technical University Dresden, Germany (S.M.-R., A.E.-A.); Institute of Experimental and Clinical Pharmacology and Toxicology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany (C.V.); Cellular Biochemistry, Department of Biology, University of Kaiserslautern, Germany (B.M.); Department of Cellular Biochemistry, University Medical Center Göttingen, Germany (S.D.); Cardiovascular Division, King's College London, British Heart Foundation Centre, United Kingdom (A.M.S.); and German Center for Cardiovascular Research, University Medical Center Hamburg-Eppendorf, Hamburg, Germany (V.O.N.); and Institute of Experimental Cardiovascular Research, Hamburg, Germany (V.O.N.).

Rationale: Changes in redox potentials of cardiac myocytes are linked to several cardiovascular diseases. Redox alterations are currently mostly described qualitatively using chemical sensors, which however do not allow quantifying redox potentials, lack specificity, and the possibility to analyze subcellular domains. Recent advances to quantitatively describe defined redox changes include the application of genetically encoded redox biosensors.

Objective: Establishment of mouse models, which allow the quantification of the glutathione redox potential (E) in the cytoplasm and the mitochondrial matrix of isolated cardiac myocytes and in Langendorff-perfused hearts based on the use of the redox-sensitive green fluorescent protein 2, coupled to the glutaredoxin 1 (Grx1-roGFP2).

Methods And Results: We generated transgenic mice with cardiac myocyte-restricted expression of Grx1-roGFP2 targeted either to the mitochondrial matrix or to the cytoplasm. The response of the roGFP2 toward HO, diamide, and dithiothreitol was titrated and used to determine the E in isolated cardiac myocytes and in Langendorff-perfused hearts. Distinct E were observed in the cytoplasm and the mitochondrial matrix. Stimulation of the cardiac myocytes with isoprenaline, angiotensin II, or exposure to hypoxia/reoxygenation additionally underscored that these compartments responded independently. A compartment-specific response was also observed 3 to 14 days after myocardial infarction.

Conclusions: We introduce redox biosensor mice as a new tool, which allows quantification of defined alterations of E in the cytoplasm and the mitochondrial matrix in cardiac myocytes and can be exploited to answer questions in basic and translational cardiovascular research.
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http://dx.doi.org/10.1161/CIRCRESAHA.116.309551DOI Listing
October 2016

Cardiomyocyte-Specific Transgenic Expression of Prolyl-4-Hydroxylase Domain 3 Impairs the Myocardial Response to Ischemia.

Cell Physiol Biochem 2015 27;36(3):843-51. Epub 2015 May 27.

Institute of Cardiovascular Physiology, University Medical Center Göttingen, Göttingen, Germany.

Aims: The prolyl-4-hydroxylase domain (PHD) enzymes are representing novel therapeutic targets for ischemic tissue protection. Whereas the consequences of a knock out of the PHDs have been analyzed in the context of cardioprotection, the implications of PHD overexpression is unknown so far.

Methods And Results: We generated cardiomyocyte-specific PHD3transgenic mice (cPhd3tg). Resting cPhd3tg mice did not show constitutive accumulation of HIF-1α or HIF-2α or changes in HIF target gene expression in the heart. Cardiac function was followed up for 14 months in these mice and found to be unchanged. After challenging the cPhd3tg mice with ligation of the left anterior descending artery, HIF-1α/-2α accumulation in the left ventricles was blunted. This was associated with a significantly increased infarct size of the cPhd3tg compared to wild type mice.

Conclusion: Whereas overexpression of PHD3 in the resting state does not significantly influence cardiac function, it is crucial for the cardiac response to ischemia by affecting HIFα accumulation in the ischemic tissue.
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http://dx.doi.org/10.1159/000430260DOI Listing
March 2016

Pre- and post-conditional inhibition of prolyl-4-hydroxylase domain enzymes protects the heart from an ischemic insult.

Pflugers Arch 2015 Oct 13;467(10):2141-9. Epub 2015 Jan 13.

Institute of Cardiovascular Physiology, University Medical Center, Georg-August-University Göttingen, Humboldtallee 23, 37073, Göttingen, Germany.

Several genetically modified mouse models implicated that prolyl-4-hydroxylase domain (PHD) enzymes are critical mediators for protecting tissues from an ischemic insult including myocardial infarction by affecting the stability and activation of hypoxia-inducible factor (HIF)-1 and HIF-2. Thus, the current efforts to develop small-molecule PHD inhibitors open a new therapeutic option for myocardial tissue protection during ischemia. Therefore, we aimed to investigate the applicability and efficacy of pharmacological HIFα stabilization by a small-molecule PHD inhibitor in the heart. We tested for protective effects in the acute phase of myocardial infarction after pre- or post-conditional application of the inhibitor. Application of the specific PHD inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) resulted in HIF-1α and HIF-2α accumulation in heart muscle cells in vitro and in vivo. The rapid and robust responsiveness of cardiac tissue towards ICA was further confirmed by induction of the known HIF target genes heme oxygenase-1 and PHD3. Pre- and post-conditional treatment of mice undergoing myocardial infarction resulted in a significantly smaller infarct size. Tissue protection from ischemia after pre- or post-conditional ICA treatment demonstrates that there is a therapeutic time window for the application of the PHD inhibitor (PHI) post-myocardial infarction, which might be exploited for acute medical interventions.
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http://dx.doi.org/10.1007/s00424-014-1667-zDOI Listing
October 2015

Lights on for HIF-1α: genetically enhanced mouse cardiomyocytes for heart tissue imaging.

Cell Physiol Biochem 2014 30;34(2):455-62. Epub 2014 Jul 30.

Institute of Cardiovascular Physiology, Georg-August-University Göttingen, Göttingen, Germany.

Background/aims: The hypoxia inducible factor-1 (HIF-1) is a suitable marker for tissue oxygenation. We intended to develop cardiomyocytes (CMs) expressing the oxygen-dependent degradation domain of HIF-1α fused to the firefly luciferase (ODD-Luc) followed by proof-of-concept for its applicability in the assessment of heart muscle oxygenation.

Methods And Results: We first generated embryonic stem cell (ESC) lines (ODD-Luc ESCs) from a Tg ROSA26 ODD-Luc/+ mouse. Subsequent CMs selection was facilitated by stable integration of an antibiotic resistance expressed under the control of the αMHC promoter. ODD-Luc ESCs showed a strong Luc-signal within 1 h of hypoxia (1% oxygen), which coincided with endogenous HIF-1α. Engineered heart muscle (EHM) constructed with ODD-Luc CMs confirmed the utility of the model to sense hypoxia, and monitor reoxygenation also in a multicellular heart muscle model. Pharmacologically induced inotropy/chronotropy under isoprenaline resulted in enhanced Luc-signal suggesting enhanced oxygen consumption, leading to notable myocardial hypoxia.

Conclusions: ODD-Luc-CMs can be used to monitor dynamic changes of cardiomyocyte oxygenation in living heart muscle samples. We provide proof-of-concept for pharmacologically induced myocardial interventions and envision applications of the developed model in drug screens and fundamental studies of ischemia/reperfusion injury.
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http://dx.doi.org/10.1159/000363014DOI Listing
April 2015

Hypoxia and the modulation of the actin cytoskeleton - emerging interrelations.

Authors:
Anke Zieseniss

Hypoxia (Auckl) 2014 25;2:11-21. Epub 2014 Mar 25.

Institute of Cardiovascular Physiology, University Medical Center, Georg-August University, Göttingen, Germany.

Recent progress in understanding the influence of hypoxia on cell function has revealed new information about the interrelationship between the actin cytoskeleton and hypoxia; nevertheless, details remain cloudy. The dynamic regulation of the actin cytoskeleton during hypoxia is complex, varies in different cells and tissues, and also depends on the mode of hypoxia. Several molecular players and pathways are emerging that contribute to the modulation of the actin cytoskeleton and that affect the large repertoire of actin-binding proteins in hypoxia. This review describes and discusses the accumulated knowledge about actin cytoskeleton dynamics in hypoxia, placing special emphasis on the Rho family of small guanosine triphosphatases (Rho GTPases). Given that RhoA, Rac and Cdc42 are very well characterized, the review is focused on these family members of Rho GTPases. Notably, in several cell types and tissues, hypoxia, presumably via Rho GTPase signaling, induces actin rearrangement and actin stress fiber assembly, which is a prevalent modulation of the actin cytoskeleton in hypoxia.
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http://dx.doi.org/10.2147/HP.S53575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045051PMC
March 2014

Hypoxia modulates fibroblastic architecture, adhesion and migration: a role for HIF-1α in cofilin regulation and cytoplasmic actin distribution.

PLoS One 2013 18;8(7):e69128. Epub 2013 Jul 18.

Institute of Cardiovascular Physiology, University Medical Center, Georg-August-University Goettingen, Goettingen, Germany.

Cells can adapt to hypoxia by various mechanisms. Yet, hypoxia-induced effects on the cytoskeleton-based cell architecture and functions are largely unknown. Here we present a comprehensive analysis of the architecture and function of L929 fibroblasts under hypoxic conditions (1% O2). Cells cultivated in hypoxia showed striking morphological differences as compared to cells cultivated under normoxic conditions (20% O2). These changes include an enlargement of cell area and volume, increased numbers of focal contacts and loss of cell polarization. Furthermore the β- and γ-actin distribution is greatly altered. These hypoxic adjustments are associated with enhanced cell spreading and a decline of cell motility in wound closure and single cell motility assays. As the hypoxia-inducible factor-1α (HIF-1α) is stabilised in hypoxia and plays a pivotal role in the transcriptional response to changes in oxygen availability we used an shRNA-approach to examine the role of HIF-1α in cytoskeleton-related architecture and functions. We show that the observed increase in cell area, actin filament rearrangement, decrease of single cell migration in hypoxia and the maintenance of p-cofilin levels is dependent on HIF-1α stabilisation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069128PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3715466PMC
February 2014

A novel mechanism involving four-and-a-half LIM domain protein-1 and extracellular signal-regulated kinase-2 regulates titin phosphorylation and mechanics.

J Biol Chem 2012 Aug 9;287(35):29273-84. Epub 2012 Jul 9.

Department of Medicine, University of California-San Diego, La Jolla, CA 92093, USA.

Understanding mechanisms underlying titin regulation in cardiac muscle function is of critical importance given recent compelling evidence that highlight titin mutations as major determinants of human cardiomyopathy. We previously identified a cardiac biomechanical stress-regulated complex at the cardiac-specific N2B region of titin that includes four-and-a-half LIM domain protein-1 (Fhl1) and components of the mitogen-activated protein signaling cascade, which impacted muscle compliance in Fhl1 knock-out cardiac muscle. However, direct regulation of these molecular components in mediating titin N2B function remained unresolved. Here we identify Fhl1 as a novel negative regulator of titin N2B levels and phosphorylation-mediated mechanics. We specifically identify titin N2B as a novel substrate of extracellular signal regulated-kinase-2 (Erk2) and demonstrate that Fhl1 directly interferes with Erk2-mediated titin-N2B phosphorylation. We highlight the critical region in titin-N2B that interacts with Fhl1 and residues that are dependent on Erk2-mediated phosphorylation in situ. We also propose a potential mechanism for a known titin-N2B cardiomyopathy-causing mutation that involves this regulatory complex. These studies shed light on a novel mechanism regulating titin-N2B mechano-signaling as well as suggest that dysfunction of these pathways could be important in cardiac disease states affecting muscle compliance.
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http://dx.doi.org/10.1074/jbc.M112.372839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436149PMC
August 2012

Rcf1 mediates cytochrome oxidase assembly and respirasome formation, revealing heterogeneity of the enzyme complex.

Cell Metab 2012 Mar 16;15(3):336-47. Epub 2012 Feb 16.

Department of Biochemistry II, University of Göttingen, D-37073 Göttingen, Germany.

The terminal enzyme of the mitochondrial respiratory chain, cytochrome oxidase, transfers electrons to molecular oxygen, generating water. Within the inner mitochondrial membrane, cytochrome oxidase assembles into supercomplexes, together with other respiratory chain complexes, forming so-called respirasomes. Little is known about how these higher oligomeric structures are attained. Here we report on Rcf1 and Rcf2 as cytochrome oxidase subunits in S. cerevisiae. While Rcf2 is specific to yeast, Rcf1 is a conserved subunit with two human orthologs, RCF1a and RCF1b. Rcf1 is required for growth in hypoxia and complex assembly of subunits Cox13 and Rcf2, as well as for the oligomerization of a subclass of cytochrome oxidase complexes into respirasomes. Our analyses reveal that the cytochrome oxidase of mitochondria displays intrinsic heterogeneity with regard to its subunit composition and that distinct forms of respirasomes can be formed by complex variants.
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http://dx.doi.org/10.1016/j.cmet.2012.01.016DOI Listing
March 2012

Unfavourable consequences of chronic cardiac HIF-1α stabilization.

Cardiovasc Res 2012 Apr 18;94(1):77-86. Epub 2012 Jan 18.

Department of Cardiovascular Physiology, Universitätsmedizin, Georg-August-University Göttingen, Humboldtallee 23, 37073 Göttingen, Germany.

Aims: The hypoxia-inducible factor-1 (HIF-1) is the master modulator of hypoxic gene expression. The effects of chronically stabilized cardiac HIF-1α and its role in the diseased heart are not precisely known. The aims of this study were as follows: (i) to elucidate consequences of HIF-1α stabilization in the heart; (ii) to analyse long-term effects of HIF-1α stabilization with ageing and the ability of the HIF-1α overexpressing hearts to respond to increased mechanical load; and (iii) to analyse HIF-1α protein levels in failing heart samples.

Methods And Results: In a cardiac-specific HIF-1α transgenic mouse model, constitutive expression of HIF-1α leads to changes in capillary area and shifts the cardiac metabolism towards glycolysis with a net increase in glucose uptake. Furthermore, Ca(2+) handling is altered, with increased Ca(2)(+) transients and faster intracellular [Ca(2+)] decline. These changes are associated with decreased expression of sarcoplasmic/endoplasmic reticulum calcium ATPase 2a but elevated phosphorylation of phospholamban. HIF-1α transgenic mice subjected to transverse aortic constriction exhibited profound cardiac decompensation. Moreover, cardiomyopathy was also seen in ageing transgenic mice. In parallel, we found an increased stabilization of HIF-1α in heart samples of patients with end-stage heart failure.

Conclusion: Changes induced with transgenic cardiac HIF-1α possibly mediate beneficial effects in the short term; however, with increased mechanical load and ageing they become detrimental for cardiac function. Together with the finding of increased HIF-1α protein levels in samples from human patients with cardiomyopathy, these data indicate that chronic HIF-1α stabilization drives autonomous pathways that add to disease progression.
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http://dx.doi.org/10.1093/cvr/cvs014DOI Listing
April 2012

Smyd2 controls cytoplasmic lysine methylation of Hsp90 and myofilament organization.

Genes Dev 2012 Jan 12;26(2):114-9. Epub 2012 Jan 12.

Laboratory of Immune Cell Epigenetics and Signaling, The Rockefeller University, New York, NY 10065, USA.

Protein lysine methylation is one of the most widespread post-translational modifications in the nuclei of eukaryotic cells. Methylated lysines on histones and nonhistone proteins promote the formation of protein complexes that control gene expression and DNA replication and repair. In the cytoplasm, however, the role of lysine methylation in protein complex formation is not well established. Here we report that the cytoplasmic protein chaperone Hsp90 is methylated by the lysine methyltransferase Smyd2 in various cell types. In muscle, Hsp90 methylation contributes to the formation of a protein complex containing Smyd2, Hsp90, and the sarcomeric protein titin. Deficiency in Smyd2 results in the loss of Hsp90 methylation, impaired titin stability, and altered muscle function. Collectively, our data reveal a cytoplasmic protein network that employs lysine methylation for the maintenance and function of skeletal muscle.
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http://dx.doi.org/10.1101/gad.177758.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3273835PMC
January 2012

Terminal differentiation, advanced organotypic maturation, and modeling of hypertrophic growth in engineered heart tissue.

Circ Res 2011 Oct 15;109(10):1105-14. Epub 2011 Sep 15.

Department of Pharmacology, Georg-August-University Goettingen, Robert-Koch-Str 40, 37075 Goettingen, Germany.

Rationale: Cardiac tissue engineering should provide "realistic" in vitro heart muscle models and surrogate tissue for myocardial repair. For either application, engineered myocardium should display features of native myocardium, including terminal differentiation, organotypic maturation, and hypertrophic growth.

Objective: To test the hypothesis that 3D-engineered heart tissue (EHT) culture supports (1) terminal differentiation as well as (2) organotypic assembly and maturation of immature cardiomyocytes, and (3) constitutes a methodological platform to investigate mechanisms underlying hypertrophic growth.

Methods And Results: We generated EHTs from neonatal rat cardiomyocytes and compared morphological and molecular properties of EHT and native myocardium from fetal, neonatal, and adult rats. We made the following key observations: cardiomyocytes in EHT (1) gained a high level of binucleation in the absence of notable cytokinesis, (2) regained a rod-shape and anisotropic sarcomere organization, (3) demonstrated a fetal-to-adult gene expression pattern, and (4) responded to distinct hypertrophic stimuli with concentric or eccentric hypertrophy and reexpression of fetal genes. The process of terminal differentiation and maturation (culture days 7-12) was preceded by a tissue consolidation phase (culture days 0-7) with substantial cardiomyocyte apoptosis and dynamic extracellular matrix restructuring.

Conclusions: This study documents the propensity of immature cardiomyocytes to terminally differentiate and mature in EHT in a remarkably organotypic manner. It moreover provides the rationale for the utility of the EHT technology as a methodological bridge between 2D cell culture and animal models.
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http://dx.doi.org/10.1161/CIRCRESAHA.111.251843DOI Listing
October 2011

Cardiomyocyte-specific prolyl-4-hydroxylase domain 2 knock out protects from acute myocardial ischemic injury.

J Biol Chem 2011 Apr 26;286(13):11185-94. Epub 2011 Jan 26.

Department of Cardiovascular Physiology, Universitätsmedizin Göttingen, Georg-August University Göttingen, Göttingen, Germany.

Prolylhydroxylase domain proteins (PHD) are cellular oxygen-sensing molecules that regulate the stability of the α-subunit of the transcription factor hypoxia inducible factor (HIF)-1. HIF-1 affects cardiac development as well as adaptation of the heart toward increased pressure overload or myocardial infarction. We have disrupted PHD2 in cardiomyocytes (cPhd (-/-)) using Phd2(flox/flox) mice in combination with MLCvCre mice, which resulted in HIF-1α stabilization and activation of HIF target genes in the heart. Although cPhd2(-/-) mice showed no gross abnormalities in cardiac filament structure or function, we observed a significant increased cardiac capillary area in those mice. cPhd2 (-/-) mice did not respond differently to increased mechanical load by transverse aortic constriction compared with their wild-type (wt) littermates. After ligation of the left anterior descending artery, however, the area at risk and area of necrosis were significantly smaller in the cPhd2(-/-) mice compared with Phd2 wt mice in line with the described pivotal role of HIF-1α for tissue protection in case of myocardial infarction. This correlated with a decreased number of apoptotic cells in the infarcted myocardium in the cPhd2(-/-) mice and significantly improved cardiac function 3 weeks after myocardial infarction.
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http://dx.doi.org/10.1074/jbc.M110.186809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064173PMC
April 2011

The Nebulin family: an actin support group.

Trends Cell Biol 2011 Jan 15;21(1):29-37. Epub 2010 Oct 15.

Department of Cell Biology, and Molecular Cardiovascular Research Program, The University of Arizona, Tucson, AZ, USA.

Nebulin, a giant, actin-binding protein, is the largest member of a family of proteins (including N-RAP, nebulette, lasp-1 and lasp-2) that are assembled in a variety of cytoskeletal structures, and expressed in different tissues. For decades, nebulin has been thought to act as a molecular ruler, specifying the precise length of actin filaments in skeletal muscle. However, emerging evidence suggests that nebulin should not be viewed as a ruler but as an actin filament stabilizer required for length maintenance. Nebulin has also been implicated recently in an array of regulatory functions independent of its role in actin filament length regulation. In this review, we discuss the current evolutionary, biochemical, and functional data for the nebulin family of proteins - a family whose members, both large and small, function as cytoskeletal scaffolds and stabilizers.
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http://dx.doi.org/10.1016/j.tcb.2010.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3014390PMC
January 2011

Prolyl hydroxylase domain (PHD) 2 affects cell migration and F-actin formation via RhoA/rho-associated kinase-dependent cofilin phosphorylation.

J Biol Chem 2010 Oct 27;285(44):33756-63. Epub 2010 Aug 27.

Department of Cardiovascular Physiology, Universitätsmedizin Göttingen, Georg-August University Göttingen, D-37073 Göttingen, Germany.

Cells are responding to hypoxia via prolyl-4-hydroxylase domain (PHD) enzymes, which are responsible for oxygen-dependent hydroxylation of the hypoxia-inducible factor (HIF)-1α subunit. To gain further insight into PHD function, we generated knockdown cell models for the PHD2 isoform, which is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. Induction of a PHD2 knockdown in tetracycline-inducible HeLa PHD2 knockdown cells resulted in increased F-actin formation as detected by phalloidin staining. A similar effect could be observed in the stably transfected PHD2 knockdown cell clones 1B6 and 3B7. F-actin is at least in part responsible for shaping cell morphology as well as regulating cell migration. Cell migration was impaired significantly as a consequence of PHD2 knockdown in a scratch assay. Mechanistically, PHD2 knockdown resulted in activation of the RhoA (Ras homolog gene family member A)/Rho-associated kinase pathway with subsequent phosphorylation of cofilin. Because cofilin phosphorylation impairs its actin-severing function, this may explain the F-actin phenotype, thereby providing a functional link between PHD2-dependent signaling and cell motility.
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http://dx.doi.org/10.1074/jbc.M110.132985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2962474PMC
October 2010

Impaired Ca(2+)-handling in HIF-1alpha(+/-) mice as a consequence of pressure overload.

Pflugers Arch 2010 Mar 8;459(4):569-77. Epub 2009 Nov 8.

Department of Cardiovascular Physiology, Heart Center Georg-August University Göttingen, Humboldtallee 23, 37073 Göttingen, Germany.

The hypoxia-inducible factor (HIF)-1 is critically involved in the cellular adaptation to a decrease in oxygen availability. The influence of HIF-1alpha for the development of cardiac hypertrophy and cardiac function that occurs in response to sustained pressure overload has been mainly attributed to a challenged cardiac angiogenesis and cardiac hypertrophy up to now. Hif-1alpha (+/+) and Hif-1alpha (+/-) mice were studied regarding left ventricular hypertrophy and cardiac function after being subjected to transverse aortic constriction (TAC). After TAC, both Hif-1alpha (+/+) and Hif-1alpha (+/-) mice developed left ventricular hypertrophy with increased posterior wall thickness, septum thickness and increased left ventricular weight to a similar extent. No significant difference in cardiac vessel density was observed between Hif-1alpha (+/+) and Hif-1alpha (+/-) mice. However, only the Hif-1alpha (+/-) mice developed severe heart failure as revealed by a significantly reduced fractional shortening mostly due to increased end-systolic left ventricular diameter. On the single cell level this correlated with reduced myocyte shortenings, decreased intracellular Ca(2+)-transients and SR-Ca(2+) content in myocytes of Hif-1a (+/-) mice. Thus, HIF-1alpha can be critically involved in the preservation of cardiac function after chronic pressure overload without affecting cardiac hypertrophy. This effect is mediated via HIF-dependent modulation of cardiac calcium handling and contractility.
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http://dx.doi.org/10.1007/s00424-009-0748-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2827795PMC
March 2010

Lasp-2 expression, localization, and ligand interactions: a new Z-disc scaffolding protein.

Cell Motil Cytoskeleton 2008 Jan;65(1):59-72

Department of Cell Biology and Anatomy, University of Arizona, Tucson, Arizona 85724, USA.

The nebulin family of actin-binding proteins plays an important role in actin filament dynamics in a variety of cells including striated muscle. We report here the identification of a new striated muscle Z-disc associated protein: lasp-2 (LIM and SH3 domain protein-2). Lasp-2 is the most recently identified member of the nebulin family. To evaluate the role of lasp-2 in striated muscle, lasp-2 gene expression and localization were studied in chick and mouse tissue, as well as in primary cultures of chick cardiac and skeletal myocytes. Lasp-2 mRNA was detected as early as chick embryonic stage 25 and lasp-2 protein was associated with developing premyofibril structures, Z-discs of mature myofibrils, focal adhesions, and intercalated discs of cultured cardiomyocytes. Expression of GFP-tagged lasp-2 deletion constructs showed that the C-terminal region of lasp-2 is important for its localization in striated muscle cells. Lasp-2 organizes actin filaments into bundles and interacts directly with the Z-disc protein alpha-actinin. These results are consistent with a function of lasp-2 as a scaffolding and actin filament organizing protein within striated muscle Z-discs.
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http://dx.doi.org/10.1002/cm.20244DOI Listing
January 2008

Raver1 is an integral component of muscle contractile elements.

Cell Tissue Res 2007 Mar 10;327(3):583-94. Epub 2006 Nov 10.

Cell Biology, Zoological Institute, Technical University of Braunschweig, Biocentre, Spielmannstrasse 7, 38092 Braunschweig, Germany.

Raver1, a ubiquitously expressed protein, was originally identified as a ligand for metavinculin, the muscle-specific isoform of the microfilament-associated protein vinculin. The protein resides primarily in the nucleus, where it colocalises and may interact with polypyrimidine-tract-binding protein, which is involved in alternative splicing processes. During skeletal muscle differentiation, raver1 translocates to the cytoplasm and eventually targets the Z-line of sarcomeres. Here, it colocalises with metavinculin, vinculin and alpha-actinin, all of which have biochemically been identified as raver1 ligands. To obtain more information about the potential role of raver1 in muscle structure and function, we have investigated its distribution and fine localisation in mouse striated and smooth muscle, by using three monoclonal antibodies that recognise epitopes in different regions of the raver1 protein. Our immunofluorescence and immunoelectron-microscopic results indicate that the cytoplasmic accumulation of raver1 is not confined to skeletal muscle but also occurs in heart and smooth muscle. Unlike vinculin and metavinculin, cytoplasmic raver1 is not restricted to costameres but additionally represents an integral part of the sarcomere. In isolated myofibrils and in ultrathin sections of skeletal muscle, raver1 has been found concentrated at the I-Z-I band. A minor fraction of raver1 is present in the nuclei of all three types of muscle. These data indicate that, during muscle differentiation, raver1 might link gene expression with structural functions of the contractile machinery of muscle.
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http://dx.doi.org/10.1007/s00441-006-0322-1DOI Listing
March 2007

Comparative biochemical analysis suggests that vinculin and metavinculin cooperate in muscular adhesion sites.

J Biol Chem 2004 Jul 24;279(30):31533-43. Epub 2004 May 24.

Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38092 Braunschweig, Germany.

Metavinculin, the muscle-specific splice variant of the cell adhesion protein vinculin, is characterized by a 68-amino acid insert within the C-terminal tail domain. The findings that mutations within this region correlate with hereditary idiopathic dilated cardiomyopathy in man suggest a specific contribution of metavinculin to the molecular architecture of muscular actin-membrane attachment sites, the nature of which, however, is still unknown. In mice, metavinculin is expressed in smooth and skeletal muscle, where it co-localizes with vinculin in dense plaques and costameres, respectively, but is of conspicuously low abundance in the heart. Immunoprecipitates suggest that both isoforms are present in the same complex. On the molecular level, both vinculin isoforms are regulated via an intramolecular head-tail interaction, with the metavinculin tail domain having a lower affinity for the head as compared with the vinculin tail. In addition, metavinculin displays impaired binding to acidic phospholipids and reduced homodimerization. Only in the presence of phospholipid-activated vinculin tail, the metavinculin tail domain is readily incorporated into heterodimers. Mutational analysis revealed that the metavinculin insert significantly alters binding of the C-terminal hairpin loop to acidic phospholipids. In summary, our data lead to a model in which unfurling of the metavinculin tail domain is impaired by the negative charges of the 68-amino acid insert, thus requiring vinculin to fully activate the metavinculin molecule. As a consequence, microfilament anchorage may be modulated at muscular adhesion sites through heterodimer formation.
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http://dx.doi.org/10.1074/jbc.M314245200DOI Listing
July 2004

A lipid-regulated docking site on vinculin for protein kinase C.

J Biol Chem 2002 Mar 10;277(9):7396-404. Epub 2001 Dec 10.

Department of Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38092 Braunschweig, Germany.

During cell spreading, binding of actin-organizing proteins to acidic phospholipids and phosphorylation are important for localization and activity of these proteins at nascent cell-matrix adhesion sites. Here, we report on a transient interaction between the lipid-dependent protein kinase Calpha and vinculin, an early component of these sites, during spreading of HeLa cells on collagen. In vitro binding of protein kinase Calpha to vinculin tail was found dependent on free calcium and acidic phospholipids but independent of a functional kinase domain. The interaction was enhanced by conditions that favor the oligomerization of vinculin. Phosphorylation by protein kinase Calpha reached 1.5 mol of phosphate/mol of vinculin tail and required the C-terminal hydrophobic hairpin, a putative phosphatidylinositol 4,5-bisphosphate-binding site. Mass spectroscopy of peptides derived from in vitro phosphorylated vinculin tail identified phosphorylation of serines 1033 and 1045. Inhibition of C-terminal phospholipid binding at the vinculin tail by mutagenesis or deletion reduced the rate of phosphorylation to < or =50%. We suggest a possible mechanism whereby phospholipid-regulated conformational changes in vinculin may lead to exposure of a docking site for protein kinase Calpha and subsequent phosphorylation of vinculin and/or vinculin interaction partners, thereby affecting the formation of cell adhesion complexes.
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http://dx.doi.org/10.1074/jbc.M110008200DOI Listing
March 2002