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Thomas Eschenhagen - One of the best experts on this subject based on the ideXlab platform.
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piezo bending actuators for isometric or auxotonic contraction analysis of Engineered Heart Tissue
Journal of Tissue Engineering and Regenerative Medicine, 2019Co-Authors: Ingra Mannhardt, Christoph Warncke, Hoc Khiem Trieu, Jorg Muller, Thomas EschenhagenAbstract:Engineered Heart Tissue (EHT) has proven as valuable tool for disease modelling, drug safety screening, and cardiac repair. Especially in combination with the stem cell technology, these in vitro models of the human Heart have generated interest not only of basic cardiovascular researchers but also of regulatory authorities responsible for drug safety. A main limitation of 3D-based assays for evaluating cardiotoxicity is their limited throughput. We integrated piezo-bending actuators in a 24-well system for the generation of strip-like rat and human EHT attached to hollow, elastic silicone posts. Muscle contractions of EHTs induced a measurable electrical current in the piezo-bending actuators that could be analysed for contraction amplitude, frequency, and contraction and relaxation kinetics. Compared with the standard video-optical analysis of contractile activity, the new system allows for (a) the analysis of several Tissues in parallel, (b) switching between auxotonic and isometric contractions by inserting a stiff metal post in the silicone post opposing the piezo actuator, (c) continuous measurement over days with low data volume (megabyte), (d) automated measurement without the necessity of adjustment of Tissue position for video-optical analysis, (e) reduced complexity and costs, (f) high sensitivity of contraction detection, (g) calculation of absolute contraction force, and (h) suitability for variable Tissue geometries. The new set-up for contraction analysis based on piezo-bending actuators is a promising new method for the parallel screening of EHT for pharmacological drug effects and other applications of muscle Tissue engineering (e.g., skeletal muscle engineering or cardiac repair).
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automated contraction analysis of human Engineered Heart Tissue for cardiac drug safety screening
Journal of Visualized Experiments, 2017Co-Authors: Ingra Mannhardt, Thomas Eschenhagen, Anika Benzin, Thomas Schulze, Umber Saleem, Birgit Klampe, Arne HansenAbstract:Cardiac Tissue engineering describes techniques to constitute three dimensional force-generating Engineered Tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate Engineered Heart Tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human Heart, indicating it to be a promising tool for cardiac drug safety screening.
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New Methods in Cardiovascular Biology Development of a Drug Screening Platform Based on Engineered Heart Tissue
2016Co-Authors: June Uebeler, Thomas EschenhagenAbstract:recent induced pluripotent stem cell technology, disease modeling, but available techniques are unsuitable for higher throughput. Objective: Here, we present a new miniaturized and automated method based on Engineered Heart Tissue (EHT). Methods and Results: Neonatal rat Heart cells are mixed with fibrinogen/Matrigel plus thrombin and pipetted into rectangular casting molds in which two flexible silicone posts are positioned from above. Contractile activity is monitored video-optically by a camera and evaluated by a custom-made software program. Fibrin-based mini-EHTs (FBMEs) (150 L, 600 000 cells) were transferred from molds to a standard 24-well plate two hours after casting. Over time FBMEs condensed from a 1233 mm gel to a muscle strip of 8 mm length and, depending on conditions, 0.2 to 1.3 mm diameter. After 8 to 10 days, FBMEs started to rhythmically deflect the posts. Post properties and the extent of post deflection allowed calculation of rate, force (0.1 to 0.3 mN), and kinetics which was validated in organ baths experiments. FBMEs exhibited a well-developed, longitudinally aligned actinin-positive cardiac muscle network and lectin-positive vascular structures interspersed homoge-neously throughout the construct. Analysis of a large series of FBME (n192) revealed high yield and reproducibility and stability for weeks. Chromanol, quinidine, and erythromycin exerted concentration-dependent increases in relaxation time, doxorubicin decreases in contractile force
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Guanabenz Interferes with ER Stress and Exerts Protective Effects in Cardiac Myocytes
2016Co-Authors: Christiane Neuber, Thomas Schulze, June Uebeler, Hannieh Sotoud, Ali El-armouche, Thomas EschenhagenAbstract:Endoplasmic reticulum (ER) stress has been implicated in a variety of cardiovascular diseases. During ER stress, disruption of the complex of protein phosphatase 1 regulatory subunit 15A and catalytic subunit of protein phosphatase 1 by the small molecule guanabenz (antihypertensive, a2-adrenoceptor agonist) and subsequent inhibition of stress-induced dephos-phorylation of eukaryotic translation initiation factor 2a (eIF2a) results in prolonged eIF2a phosphorylation, inhibition of protein synthesis and protection from ER stress. In this study we assessed whether guanabenz protects against ER stress in cardiac myocytes and affects the function of 3 dimensional Engineered Heart Tissue (EHT). We utilized neonatal rat cardiac myocytes for the assessment of cell viability and activation of ER stress-signalling pathways and EHT for functional analysis. (i) Tunicamycin induced ER stress as measured by increased mRNA and protein levels of glucose-regulated protein 78 kDa, P-eIF2a, activating transcription factor 4, C/EBP homologous protein, and cell death. (ii) Guanabenz had no measurable effect alone, but antagonized the effects of tunicamycin on ER stress markers. (iii) Tunicamycin and other known inducers of ER stress (hydrogen peroxide, doxorubicin, thapsigargin) induced cardiac myocyte death, and this was antagonized by guanabenz in a concentration- and time-dependent manner. (iv) ER stressors also induced acute or delayed contractile dysfunction in spontaneously beating EHTs and this was, with the notable exception of relaxation deficits under thapsigargin, not significantly affected by guanabenz. The data confirm that guanabenz interferes with ER stress-signallin
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Human Engineered Heart Tissue as a Versatile Tool in Basic Research and Preclinical Toxicology
2016Co-Authors: Sebastian Schaaf, Wolfram-hubertus Zimmermann, Aya Shibamiya, Marc N Hirt, Marco Mewe, L Conradi, Ra Eder, Thomas Rau, Thomas EschenhagenAbstract:Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human Engineered Heart Tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30–40% a-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5–10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 mN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the b-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for hear
Arne Hansen - One of the best experts on this subject based on the ideXlab platform.
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hypertrophic signaling compensates for contractile and metabolic consequences of dna methyltransferase 3a loss in human cardiomyocytes
Journal of Molecular and Cellular Cardiology, 2021Co-Authors: Alexandra Madsen, Arne Hansen, Marc N Hirt, Julia Krause, Grit Hoppner, Wilson Lek Wen Tan, Ives Yubin Lim, Viacheslav O Nikolaev, Roger FooAbstract:Abstract The role of DNA methylation in cardiomyocyte physiology and cardiac disease remains a matter of controversy. We have recently provided evidence for an important role of DNMT3A in human cardiomyocyte cell homeostasis and metabolism, using Engineered Heart Tissue (EHT) generated from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes carrying a knockout of the de novo DNA methyltransferase DNMT3A. Unlike isogenic control EHT, knockout EHT displayed morphological abnormalities such as lipid accumulations inside cardiomyocytes associated with impaired mitochondrial metabolism, as well as functional defects and impaired glucose metabolism. Here, we analyzed the role of DNMT3A in the setting of cardiac hypertrophy. We induced hypertrophic signaling by treatment with 50 nM endothelin-1 and 20 μM phenylephrine for one week and assessed EHT contractility, morphology, DNA methylation, and gene expression. While both knockout EHTs and isogenic controls showed the expected activation of the hypertrophic gene program, knockout EHTs were protected from hypertrophy-related functional impairment. Conversely, hypertrophic treatment prevented the metabolic consequences of a loss of DNMT3A, i.e. abolished lipid accumulation in cardiomyocytes likely by partial normalization of mitochondrial metabolism and restored glucose metabolism and metabolism-related gene expression of knockout EHT. Together, these data suggest an important role of DNA methylation not only for cardiomyocyte physiology, but also in the setting of cardiac disease.
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chronic intermittent tachypacing by an optogenetic approach induces arrhythmia vulnerability in human Engineered Heart Tissue
Cardiovascular Research, 2020Co-Authors: Marta Lemme, Bärbel Ulmer, Maksymilian Prondzynski, Bulent Aksehirlioglu, Ingke Braren, Mirja L Schulze, Djemail Ismaili, Christian Meyer, Arne HansenAbstract:AIMS Chronic tachypacing is commonly used in animals to induce cardiac dysfunction and to study mechanisms of Heart failure and arrhythmogenesis. Human induced pluripotent stem cells (hiPSC) may replace animal models to overcome species differences and ethical problems. Here, 3D Engineered Heart Tissue (EHT) was used to investigate the effect of chronic tachypacing on hiPSC-cardiomyocytes (hiPSC-CMs). METHODS AND RESULTS To avoid cell toxicity by electrical pacing, we developed an optogenetic approach. EHTs were transduced with lentivirus expressing channelrhodopsin-2 (H134R) and stimulated by 15 s bursts of blue light pulses (0.3 mW/mm2, 30 ms, 3 Hz) separated by 15 s without pacing for 3 weeks. Chronic optical tachypacing did not affect contractile peak force, but induced faster contraction kinetics, shorter action potentials, and shorter effective refractory periods. This electrical remodelling increased vulnerability to tachycardia episodes upon electrical burst pacing. Lower calsequestrin 2 protein levels, faster diastolic depolarization (DD) and efficacy of JTV-519 (46% at 1 µmol/L) to terminate tachycardia indicate alterations of Ca2+ handling being part of the underlying mechanism. However, other antiarrhythmic compounds like flecainide (69% at 1 µmol/L) and E-4031 (100% at 1 µmol/L) were also effective, but not ivabradine (1 µmol/L) or SEA0400 (10 µmol/L). CONCLUSION We demonstrated a high vulnerability to tachycardia of optically tachypaced hiPSC-CMs in EHT and the effective termination by ryanodine receptor stabilization, sodium or hERG potassium channel inhibition. This new model might serve as a preclinical tool to test antiarrhythmic drugs increasing the insight in treating ventricular tachycardia.
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atrial like Engineered Heart Tissue an in vitro model of the human atrium
Stem cell reports, 2018Co-Authors: Marta Lemme, Bärbel Ulmer, Marc D Lemoine, Frederik Flenner, Antonia T.l. Zech, Ursula Ravens, Hermann Reichenspurner, Miriam Rolgarcia, Godfrey L Smith, Arne HansenAbstract:Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are under investigation for their suitability as human models in preclinical drug development. Antiarrhythmic drug development focuses on atrial biology for the treatment of atrial fibrillation. Here we used recent retinoic acid-based protocols to generate atrial CMs from hiPSCs and establish right atrial Engineered Heart Tissue (RA-EHT) as a 3D model of human atrium. EHT from standard protocol-derived hiPSC-CMs (Ctrl-EHT) and intact human muscle strips served as comparators. RA-EHT exhibited higher mRNA and protein concentrations of atrial-selective markers, faster contraction kinetics, lower force generation, shorter action potential duration, and higher repolarization fraction than Ctrl-EHTs. In addition, RA-EHTs but not Ctrl-EHTs responded to pharmacological manipulation of atrial-selective potassium currents. RA- and Ctrl-EHTs’ behavior reflected differences between human atrial and ventricular muscle preparations. Taken together, RA-EHT is a model of human atrium that may be useful in preclinical drug screening.
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low resting membrane potential and low inward rectifier potassium currents are not inherent features of hipsc derived cardiomyocytes
Stem cell reports, 2018Co-Authors: Christiane Neuber, K. Breckwoldt, Ingra Mannhardt, Marc D Lemoine, Frederik Flenner, Andras Horvath, Alexandra Loser, Ahmet Umur Uzun, Arne HansenAbstract:Summary Human induced pluripotent stem cell (hiPSC) cardiomyocytes (CMs) show less negative resting membrane potential (RMP), which is attributed to small inward rectifier currents (I K1 ). Here, I K1 was measured in hiPSC-CMs (proprietary and commercial cell line) cultured as monolayer (ML) or 3D Engineered Heart Tissue (EHT) and, for direct comparison, in CMs from human right atrial (RA) and left ventricular (LV) Tissue. RMP was measured in isolated cells and intact Tissues. I K1 density in ML- and EHT-CMs from the proprietary line was similar to LV and RA, respectively. I K1 density in EHT-CMs from the commercial line was 2-fold smaller than in the proprietary line. RMP in EHT of both lines was similar to RA and LV. Repolarization fraction and I K,ACh response discriminated best between RA and LV and indicated predominantly ventricular phenotype in hiPSC-CMs/EHT. The data indicate that I K1 is not necessarily low in hiPSC-CMs, and technical issues may underlie low RMP in hiPSC-CMs.
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Atrial-like Engineered Heart Tissue: An In Vitro Model of the Human Atrium
Elsevier, 2018Co-Authors: Marta Lemme, Marc D Lemoine, Frederik Flenner, Bärbel M. Ulmer, Antonia T.l. Zech, Ursula Ravens, Hermann Reichenspurner, Miriam Rol-garcia, Godfrey Smith, Arne HansenAbstract:Summary: Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are under investigation for their suitability as human models in preclinical drug development. Antiarrhythmic drug development focuses on atrial biology for the treatment of atrial fibrillation. Here we used recent retinoic acid-based protocols to generate atrial CMs from hiPSCs and establish right atrial Engineered Heart Tissue (RA-EHT) as a 3D model of human atrium. EHT from standard protocol-derived hiPSC-CMs (Ctrl-EHT) and intact human muscle strips served as comparators. RA-EHT exhibited higher mRNA and protein concentrations of atrial-selective markers, faster contraction kinetics, lower force generation, shorter action potential duration, and higher repolarization fraction than Ctrl-EHTs. In addition, RA-EHTs but not Ctrl-EHTs responded to pharmacological manipulation of atrial-selective potassium currents. RA- and Ctrl-EHTs’ behavior reflected differences between human atrial and ventricular muscle preparations. Taken together, RA-EHT is a model of human atrium that may be useful in preclinical drug screening. : Lemme et al. developed a human, atrial-like Engineered Heart Tissue from hiPSCs that could be used as an in vitro model of the human atrium to evaluate selectivity of novel ion channel blockers for atrial fibrillation. Keywords: hiPSC-CMs, pluripotent stem cells, atrial differentiation, atrial myocytes, atrial-like cells, retinoic acid, Engineered Heart Tissue, cardiac Tissue engineering, atrial fibrillatio
Alexandra Eder - One of the best experts on this subject based on the ideXlab platform.
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human Engineered Heart Tissue as a model system for drug testing
Advanced Drug Delivery Reviews, 2016Co-Authors: Alexandra Eder, Ingra Vollert, Arne Hansen, Thomas EschenhagenAbstract:Drug development is time- and cost-intensive and, despite extensive efforts, still hampered by the limited value of current preclinical test systems to predict side effects, including proarrhythmic and cardiotoxic effects in clinical practice. Part of the problem may be related to species-dependent differences in cardiomyocyte biology. Therefore, the event of readily available human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) has raised hopes that this human test bed could improve preclinical safety pharmacology as well as drug discovery approaches. However, hiPSC-CM are immature and exhibit peculiarities in terms of ion channel function, gene expression, structural organization and functional responses to drugs that limit their present usefulness. Current efforts are thus directed towards improving hiPSC-CM maturity and high-content readouts. Culturing hiPSC-CM as 3-dimensional Engineered Heart Tissue (EHT) improves CM maturity and anisotropy and, in a 24-well format using silicone racks, enables automated, multiplexed high content readout of contractile function. This review summarizes the principal technology and focuses on advantages and disadvantages of this technology and its potential for preclinical drug screening.
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Human Engineered Heart Tissue: Analysis of Contractile Force
Stem Cell Reports, 2016Co-Authors: Ingra Mannhardt, Sebastian Schaaf, Christiane Neuber, David Letuffe-brenière, K. Breckwoldt, Alexandra Eder, Herbert Schulz, Anika Benzin, Tessa Werner, Thomas SchulzeAbstract:Analyzing contractile force, the most important and best understood function of cardiomyocytes in vivo is not established in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). This study describes the generation of 3D, strip-format, force-generating Engineered Heart Tissues (EHT) from hiPSC-CM and their physiological and pharmacological properties. CM were differentiated from hiPSC by a growth factor-based three-stage protocol. EHTs were generated and analyzed histologically and functionally. HiPSC-CM in EHTs showed well-developed sarcomeric organization and alignment, and frequent mitochondria. Systematic contractility analysis (26 concentration-response curves) reveals that EHTs replicated canonical response to physiological and pharmacological regulators of inotropy, membrane- and calcium-clock mediators of pacemaking, modulators of ion-channel currents, and proarrhythmic compounds with unprecedented precision. The analysis demonstrates a high degree of similarity between hiPSC-CM in EHT format and native human Heart Tissue, indicating that human EHTs are useful for preclinical drug testing and disease modeling.
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The concept of a subpulmonary „neo-ventricle”from Engineered Heart Tissue.
2016Co-Authors: Daniel Biermann, Alexandra Eder, Hermann Reichenspurner, Florian Arndt, Hatim Seoudy, Thomas Mir, Arlindo Riso, Rainer Kozlik-feldmann, Kersten Peldschus, Michael G. KaulAbstract:A: Extracardiac tunnel for the treatment of children with univentricular Hearts. Commonly, a non-contractile GoreTex-conduit is used to bypass blood from the inferior caval vein to the right pulmonary artery. B: Our group aims for a contractile, valved conduit made from Engineered Heart Tissue (EHT) to propel blood actively through the lungs and avoid endorgan damage in the long-term.
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automated analysis of contractile force and ca2 transients in Engineered Heart Tissue
American Journal of Physiology-heart and Circulatory Physiology, 2014Co-Authors: Andrea Stoehr, Ingra Vollert, Sebastian Schaaf, Christiane Neuber, Alexandra Eder, Frederik Flenner, Lucie Carrier, Christina Baldauf, Felix W Friedrich, Marc N HirtAbstract:Contraction and relaxation are fundamental aspects of cardiomyocyte functional biology. They reflect the response of the contractile machinery to the systolic increase and diastolic decrease of the cytoplasmic Ca2+ concentration. The analysis of contractile function and Ca2+ transients is therefore important to discriminate between myofilament responsiveness and changes in Ca2+ homeostasis. This article describes an automated technology to perform sequential analysis of contractile force and Ca2+ transients in up to 11 strip-format, fibrin-based rat, mouse, and human fura-2-loaded Engineered Heart Tissues (EHTs) under perfusion and electrical stimulation. Measurements in EHTs under increasing concentrations of extracellular Ca2+ and responses to isoprenaline and carbachol demonstrate that EHTs recapitulate basic principles of Heart Tissue functional biology. Ca2+ concentration-response curves in rat, mouse, and human EHTs indicated different maximal twitch forces (0.22, 0.05, and 0.08 mN in rat, mouse, and human, respectively; P < 0.001) and different sensitivity to external Ca2+ (EC50: 0.15, 0.39, and 1.05 mM Ca2+ in rat, mouse, and human, respectively; P < 0.001) in the three groups. In contrast, no difference in myofilament Ca2+ sensitivity was detected between skinned rat and human EHTs, suggesting that the difference in sensitivity to external Ca2+ concentration is due to changes in Ca2+ handling proteins. Finally, this study confirms that fura-2 has Ca2+ buffering effects and is thereby changing the force response to extracellular Ca2+.
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in vitro perfusion of Engineered Heart Tissue through endothelialized channels
Tissue Engineering Part A, 2013Co-Authors: Ingra Vollert, Alexandra Eder, Thomas Schulze, June Uebeler, L Conradi, Moritz Seiffert, Johanna Bachmair, Merle Sander, Alexander Vogelsang, Wolfgang HolnthonerAbstract:In Engineered Heart Tissues (EHT), oxygen and nutrient supply via mere diffusion is a likely factor limiting the thickness of cardiac muscle strands. Here, we report on a novel method to in vitro perfuse EHT through tubular channels. Adapting our previously published protocols, we expanded a miniaturized fibrin-based EHT-format to a larger six-well format with six flexible silicone posts holding each EHT (15×25×3 mm3). Thin dry alginate fibers (17×0.04×0.04 mm) were embedded into the cell–fibrin–thrombin mix and, after fibrin polymerization, dissolved by incubation in alginate lyase or sodium citrate. Oxygen concentrations were measured with a microsensor in 14-day-old EHTs (37°C, 21% oxygen) and ranged between 9% at the edges and 2% in the center of the Tissue. Perfusion rapidly increased it to 10%–12% in the immediate vicinity of the microchannel. Continuous perfusion (20 μL/h, for 3 weeks) of the tubular lumina (100–500 μm) via hollow posts of the silicone rack increased mean dystrophin-positive cardio...
Marc N Hirt - One of the best experts on this subject based on the ideXlab platform.
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hypertrophic signaling compensates for contractile and metabolic consequences of dna methyltransferase 3a loss in human cardiomyocytes
Journal of Molecular and Cellular Cardiology, 2021Co-Authors: Alexandra Madsen, Arne Hansen, Marc N Hirt, Julia Krause, Grit Hoppner, Wilson Lek Wen Tan, Ives Yubin Lim, Viacheslav O Nikolaev, Roger FooAbstract:Abstract The role of DNA methylation in cardiomyocyte physiology and cardiac disease remains a matter of controversy. We have recently provided evidence for an important role of DNMT3A in human cardiomyocyte cell homeostasis and metabolism, using Engineered Heart Tissue (EHT) generated from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes carrying a knockout of the de novo DNA methyltransferase DNMT3A. Unlike isogenic control EHT, knockout EHT displayed morphological abnormalities such as lipid accumulations inside cardiomyocytes associated with impaired mitochondrial metabolism, as well as functional defects and impaired glucose metabolism. Here, we analyzed the role of DNMT3A in the setting of cardiac hypertrophy. We induced hypertrophic signaling by treatment with 50 nM endothelin-1 and 20 μM phenylephrine for one week and assessed EHT contractility, morphology, DNA methylation, and gene expression. While both knockout EHTs and isogenic controls showed the expected activation of the hypertrophic gene program, knockout EHTs were protected from hypertrophy-related functional impairment. Conversely, hypertrophic treatment prevented the metabolic consequences of a loss of DNMT3A, i.e. abolished lipid accumulation in cardiomyocytes likely by partial normalization of mitochondrial metabolism and restored glucose metabolism and metabolism-related gene expression of knockout EHT. Together, these data suggest an important role of DNA methylation not only for cardiomyocyte physiology, but also in the setting of cardiac disease.
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Image_1_S100A4 as a Target of the E3-Ligase Asb2β and Its Effect on Engineered Heart Tissue.JPEG
2018Co-Authors: Simon Braumann, Marc N Hirt, Lucie Carrier, Tilo Thottakara, Sabrina Stücker, Silke Reischmann-düsener, Elisabeth Krämer, Julia Groß, Shirin Doroudgar, Felix W FriedrichAbstract:Background: S100A4 has recently emerged as an important player in cardiac disease, affecting phenotype development in animal models of myocardial infarction and pathological cardiac hypertrophy, albeit it is unclear whether S100A4 exerts a detrimental or beneficial function. The goal of the current study was to analyze S100A4 expression in models of cardiac pathology, investigate its degradation by the ubiquitin-proteasome system (UPS), and furthermore examine the functional effects of S100A4 levels in a 3D model of Engineered Heart Tissue (EHT).Methods and Results: S100A4 mRNA and protein levels were analyzed in different models of cardiac pathology via quantitative RT-PCR and Western blot, showing a higher S100A4 steady-state protein concentration in Hearts of Mybpc3-knock-in (KI) hypertrophic cardiomyopathy (HCM) mice. COS-7 cells co-transfected with plasmids encoding mutant (MUT) Asb2β lacking the E3 ligase activity in combination with V5-tagged S100A4 plasmid presented higher S100A4-V5 protein steady-state concentrations than cells co-transfected with the Asb2β wild type (WT) plasmid. This effect was blunted by treatment with the specific proteasome inhibitor epoxomicin. Adeno-associated virus serotype 6 (AAV6)-mediated S100A4 overexpression in a 3D model of EHT did not affect contractile parameters. Immunofluorescence analysis showed a cytosolic and partly nuclear expression pattern of S100A4. Gene expression analysis in EHTs overexpressing S100A4-V5 showed markedly lower steady-state concentrations of genes involved in cardiac fibrosis and pathological cardiac hypertrophy.Conclusion: We showed that S100A4 protein level is higher in cardiac Tissue of Mybpc3-KI HCM mice probably as a result of a lower degradation by the E3 ligase Asb2β. While an overexpression of S100A4 did not alter contractile parameters in EHTs, downstream gene expression analysis points toward modulation of signaling cascades involved in fibrosis and hypertrophy.
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human ipsc derived cardiomyocytes cultured in 3d Engineered Heart Tissue show physiological upstroke velocity and sodium current density
Scientific Reports, 2017Co-Authors: Marc D Lemoine, Christiane Neuber, K. Breckwoldt, Ingra Mannhardt, Bärbel Ulmer, Maksymilian Prondzynski, Frederik Flenner, Marc N Hirt, Andras HorvathAbstract:Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising tool for drug testing and modelling genetic disorders. Abnormally low upstroke velocity is a current limitation. Here we investigated the use of 3D Engineered Heart Tissue (EHT) as a culture method with greater resemblance to human Heart Tissue in comparison to standard technique of 2D monolayer (ML) format. INa was measured in ML or EHT using the standard patch-clamp technique. INa density was ~1.8 fold larger in EHT (−18.5 ± 1.9 pA/pF; n = 17) than in ML (−10.3 ± 1.2 pA/pF; n = 23; p < 0.001), approaching densities reported for human CM. Inactivation kinetics, voltage dependency of steady-state inactivation and activation of INa did not differ between EHT and ML and were similar to previously reported values for human CM. Action potential recordings with sharp microelectrodes showed similar upstroke velocities in EHT (219 ± 15 V/s, n = 13) and human left ventricle Tissue (LV, 253 ± 7 V/s, n = 25). EHT showed a greater resemblance to LV in CM morphology and subcellular NaV1.5 distribution. INa in hiPSC-CM showed similar biophysical properties as in human CM. The EHT format promotes INa density and action potential upstroke velocity of hiPSC-CM towards adult values, indicating its usefulness as a model for excitability of human cardiac Tissue.
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Human Engineered Heart Tissue as a Versatile Tool in Basic Research and Preclinical Toxicology
2016Co-Authors: Sebastian Schaaf, Wolfram-hubertus Zimmermann, Aya Shibamiya, Marc N Hirt, Marco Mewe, L Conradi, Ra Eder, Thomas Rau, Thomas EschenhagenAbstract:Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human Engineered Heart Tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30–40% a-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5–10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 mN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the b-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for hear
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deciphering the microrna signature of pathological cardiac hypertrophy by Engineered Heart Tissue and sequencing technology
Journal of Molecular and Cellular Cardiology, 2015Co-Authors: Marc N Hirt, Tessa Werner, Jutta Starbatty, Justus Stenzig, Daniela Indenbirken, Malik Alawi, Paul Demin, Anncathrin Kunze, Arne HansenAbstract:Pathological cardiac hypertrophy and fibrosis are modulated by a set of microRNAs, most of which have been detected in biologically complex animal models of hypertrophy by arrays with moderate sensitivity and disregard of passenger strand (previously "star") microRNAs. Here, we aimed at precisely analyzing the microRNA signature of cardiac hypertrophy and fibrosis by RNA sequencing in a standardized in vitro hypertrophy model based on Engineered Heart Tissue (EHT). Spontaneously beating, force-generating fibrin EHTs from neonatal rat Heart cells were subjected to afterload enhancement for 7days (AE-EHT), and EHTs without intervention served as controls. AE resulted in reduced contractile force and relaxation velocity, fibrotic changes and reactivation of the fetal gene program. Small RNAs were extracted from control and AE-EHTs and sequencing yielded almost 750 different mature microRNAs, many of which have never been described before in rats. The detection of both arms of the precursor stem-loop (pre-miRNA), namely -3p and -5p miRs, was frequent. 22 abundantly sequenced microRNAs were >1.3× upregulated and 15 abundantly sequenced microRNAs downregulated to <0.77×. Among the upregulated microRNAs were 3 pairs of guide and passenger strand microRNAs (miR-21-5p/-3p, miR-322-5p/-3p, miR-210-3p/-5p) and one single passenger strand microRNA (miR-140-3p). Among downregulated microRNAs were 3 pairs (miR-133a-3p/-5p, miR-30e-5p/3p, miR-30c-5p/-3p). Preincubating EHTs with anti-miR-21-5p markedly attenuated the AE-induced contractile failure, cardiomyocyte hypertrophy and fibrotic response, recapitulating prior results in whole animals. Taken together, AE-induced pathological hypertrophy in EHTs is associated with 37 differentially regulated microRNAs, including many passenger strands. Antagonizing miR-21-5p ameliorates dysfunction in this model.
Haodong Chen - One of the best experts on this subject based on the ideXlab platform.
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transcriptome analysis of non human primate induced pluripotent stem cell derived cardiomyocytes in 2d monolayer culture vs 3d Engineered Heart Tissue
Cardiovascular Research, 2021Co-Authors: Huaxiao Yang, Ningyi Shao, Alexandra Holmstrom, Xin Zhao, Tony Chour, Haodong ChenAbstract:Aims Stem cell therapy has shown promise for treating myocardial infarction via re-muscularization and paracrine signalling in both small and large animals. Non-human primates (NHPs), such as rhesus macaques (Macaca mulatta), are primarily utilized in preclinical trials due to their similarity to humans, both genetically and physiologically. Currently, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are delivered into the infarcted myocardium by either direct cell injection or an Engineered Tissue patch. Although both approaches have advantages in terms of sample preparation, cell-host interaction, and engraftment, how the iPSC-CMs respond to ischaemic conditions in the infarcted Heart under these two different delivery approaches remains unclear. Here, we aim to gain a better understanding of the effects of hypoxia on iPSC-CMs at the transcriptome level. Methods and results NHP iPSC-CMs in both monolayer culture (2D) and Engineered Heart Tissue (EHT) (3D) format were exposed to hypoxic conditions to serve as surrogates of direct cell injection and Tissue implantation in vivo, respectively. Outcomes were compared at the transcriptome level. We found the 3D EHT model was more sensitive to ischaemic conditions and similar to the native in vivo myocardium in terms of cell-extracellular matrix/cell-cell interactions, energy metabolism, and paracrine signalling. Conclusion By exposing NHP iPSC-CMs to different culture conditions, transcriptome profiling improves our understanding of the mechanism of ischaemic injury.
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transcriptome analysis of non human primate induced pluripotent stem cell derived cardiomyocytes in 2d monolayer culture versus 3d Engineered Heart Tissue
Cardiovascular Research, 2020Co-Authors: Huaxiao Yang, Ningyi Shao, Alexandra Holmstrom, Xin Zhao, Tony Chour, Haodong ChenAbstract:Aims Stem cell therapy has shown promise for treating myocardial infarction (MI) via re-muscularization and paracrine signaling in both small and large animals. Non-human primates (NHPs), such as rhesus macaques (Macaca mulatta), are primarily utilized in preclinical trials due to their similarity to humans, both genetically and physiologically. Currently, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are delivered into the infarcted myocardium by either direct cell injection or an Engineered Tissue patch. Although both approaches have advantages in terms of sample preparation, cell-host interaction, and engraftment, how the iPSC-CMs respond to ischemic conditions in the infarcted Heart under these two different delivery approaches remain unclear. Here we aim to gain a better understanding of the effects of hypoxia on iPSC-CMs at the transcriptome level. Methods and results NHP iPSC-CMs in both monolayer culture (2 D) and Engineered Heart Tissue (EHT) (3 D) format were exposed to hypoxic conditions to serve as surrogates of direct cell injection and Tissue implantation in vivo, respectively. Outcomes were compared at the transcriptome level. We found the 3 D EHT model was more sensitive to ischemic conditions and similar to the native in vivo myocardium in terms of cell-extracellular matrix/cell-cell interactions, energy metabolism, and paracrine signaling. Conclusions By exposing NHP iPSC-CMs to different culture conditions, transcriptome profiling improves our understanding of the mechanism of ischemic injury. Translational perspective Stem cell therapy has shown promise for treating ischemic Heart Tissue. However, how stem cells respond following different delivery method is unclear. Here hypoxic conditioning was applied to non-human primate iPSC-CMs in 2 D monolayer culture and 3 D Engineered Heart Tissue to model cell injection versus patch implantation, respectively, in an ischemic milieu. The differential transcriptome of hypoxic effects on iPSC-CMs show upregulation of ECM-cell/cell-cell interactions (COL9A1, ITGB6, CTSV, and EPHA1), energy metabolism/hypoxia (ALDOC, ENO2, PFKFB4, CA3, and CA9), and paracrine signaling (WNT, PDGF, FGFR, EGFR, PI3K, and VEGF) in the 3 D format, which suggest Engineered Heart Tissue as more suitable model for evaluating cardiac regenerative therapy.