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Rat atrial engineered heart tissue: a new in vitro model to study atrial biology

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Abstract

Engineered heart tissue (EHT) from rat cells is a useful tool to study ventricular biology and cardiac drug safety. Since atrial and ventricular cells differ significantly, EHT and other 3D cell culture formats generated from ventricular cells have been of limited value to study atrial biology. To date, reliable in vitro models that reflect atrial physiology are lacking. Therefore, we established a novel EHT model using rat atrial cells (atrial EHT, aEHT) to assess atrial physiology, contractility and drug response. The tissue constructs were characterized with regard to gene expression, histology, electrophysiology, and the response to atrial-specific drugs. We observed typical functional properties of atrial tissue in our model such as more regular spontaneous beating with lower force, shorter action potential duration, and faster contraction and relaxation compared to ventricular EHT (vEHT). The expression of atrial-specific genes and proteins was high, whereas ventricle-specific transcripts were virtually absent. The atrial-selective drug carbachol had a strong negative inotropic and chronotropic effect on aEHT only. Taken together, the results demonstrate the feasibility of aEHT as a novel atrial 3D model and as a benchmark for tissue engineering with human induced pluripotent stem cell-derived atrial-like cardiomyocytes. Atrial EHT faithfully recapitulates atrial physiology and shall be useful to study atrial molecular physiology in health and disease as well as drug response.

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Acknowledgements

The authors want to thank Klaus Söhren, June Uebeler, Thomas Schulze, Tim Hartmann, and Grit Höppner for expert technical assistance. We additionally thank Tobias Krause for help with electrophysiological characterization, Alexander Fischer for imaging, Kristin Hartmann at Mouse Pathology Core Facility in Hamburg for histological staining, the team at FACS-Sorting Core Facility in Hamburg for their service and help regarding flow cytometry, and the UKE Microscopy Imaging Facility.

Funding

This work was supported by funding from the DZHK (German Centre for Cardiovascular Research), the German Ministry of Education and Research (BMBF) and the e:Med symAtrial consortium, a BMBF initiative.

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Author notes

  1. Julia Krause and Alexandra Löser contributed equally.

Authors and Affiliations

  1. Department of General and Interventional Cardiology, University Heart Center Hamburg, Martinistrasse 52, 20246, Hamburg, Germany

    Julia Krause, Stefan Blankenberg & Tanja Zeller

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

    Julia Krause, Alexandra Löser, Marc D. Lemoine, Torsten Christ, Katharina Scherschel, Christian Meyer, Stefan Blankenberg, Tanja Zeller, Thomas Eschenhagen & Justus Stenzig

  3. Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany

    Alexandra Löser, Marc D. Lemoine, Torsten Christ, Thomas Eschenhagen & Justus Stenzig

  4. Department of Cardiology-Electrophysiology, University Heart Center Hamburg, Martinistrasse 52, 20246, Hamburg, Germany

    Marc D. Lemoine, Katharina Scherschel & Christian Meyer

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  1. Julia Krause
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Contributions

All authors participated in the design of the experiments. JK performed atrial cell isolation and EHT preparation. AL performed force frequency experiments and histology. JK and AL performed drug response experiments, gene expression analysis, and flow cytometry. Contractility was assessed by JK, AL and JS. JK and KS performed whole-mount immunostainings. All electrophysiological experiments were done by MDL, with help from Tobias Krause and TC. Rat contractility data was contributed by Klaus Söhren and TC. JK, AL, JS and TE wrote the manuscript.

Corresponding author

Correspondence to Justus Stenzig.

Ethics declarations

Ethical standards

All animal work was conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the United States National Institutes of Health (NIH publication No. 85-23, revised 1996). All animal work was approved by the local Animal Welfare Committee of the City of Hamburg, Germany (approval #08/14). The manuscript does not contain clinical studies or patient data.

Conflict of interest

TE is cofounder of EHT Technologies GmbH Hamburg, which provides technical equipment for making and video-optical analysis of EHTs.

Electronic supplementary material

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Fig. S1 Optimization of protocols.

Flow cytometric analysis of cell quality after both atrial and ventricular cell preparation. a) Cell viability was analyzed by staining with the fixable viability dye eFluor450. b) Cardiomyocyte content was determined by staining for cardiac troponin T. c) Effect of mitosis inhibition on EHT culture. Comparison of one-time only and weekly treatment with AraC with regard to d) cultivation duration and e) stability of resting length. f) Relative transcript levels of fibroblast marker genes after culture period for verification of AraC effect on fibroblast proliferation. Transcript levels were normalized to Gusb transcript levels. Mean ± SEM, unpaired t-test, ***p<0.001, numbers in brackets refer to total number of EHTs/number of batches. Fig. S2 EHT morphology. a) and b) representative H&E staining of vEHT and aEHT paraffin sections. c) Alpha-actin stained aEHT paraffin section, shown in higher magnification in d). Fig. S3 Expression analysis of cell-type markers. Overview of all analyzed atrial-specific a) – d), ventricular-specific e) – j), and pacemaker genes k) - l). For Pitx2 (c) transcript levels were measured in left and right atria separately (LA and RA, respectively) to show chamber-specific expression. Error bars show mean ± SEM, EHT numbers refer to total number of EHTs/number of batches. Fig. S4 Batch-to-batch variability. Comparison of five vEHT and seven aEHT batches at day 14-15 with regard to a) frequency, b) force generation, c) contraction time, and d) relaxation time. Error bars show mean ± SEM, no significant differences between the batches. Fig. S5 Protocol overview. Graphical illustration of the aEHT preparation protocol including approximate time needed for each step. Tab. S1 Primer sequences. Overview of all genes analyzed with qPCR with according primer sequences. Tab. S2 Cell type-specific markers. Markers were chosen for gene expression analysis in the atrial and ventricular EHT model. (DOCX 2080 kb)

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Krause, J., Löser, A., Lemoine, M.D. et al. Rat atrial engineered heart tissue: a new in vitro model to study atrial biology. Basic Res Cardiol 113, 41 (2018). https://doi.org/10.1007/s00395-018-0701-2

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