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Phosphorylation of vasodilator-stimulated phosphoprotein contributes to myocardial ischemic preconditioning

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Abstract

Ischemic preconditioning (IP) is a well-known strategy to protect organs against cell death following ischemia. The previous work has shown that vasodilator-stimulated phosphoprotein (VASP) is involved in cytoskeletal reorganization and that it holds significant importance for the extent of myocardial ischemia reperfusion injury. Yet, the role of VASP during myocardial IP is, to date, not known. We report here that VASP phosphorylation at serine157 and serine239 is induced during hypoxia in vitro and during IP in vivo. The preconditioning-induced VASP phosphorylation inactivates the GP IIb/IIIa integrin receptor on platelets, which results in the reduced formation of organ compromising platelet neutrophil complexes. Experiments in chimeric mice confirmed the importance of VASP phosphorylation during myocardial IP. When studying this in VASP−/− animals and in an isolated heart model, we were able to confirm the important role of VASP on myocardial IP. In conclusion, we were able to show that IP-induced VASP phosphorylation in platelets is a protective mechanism against the deleterious effects of ischemia.

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Funding

This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG): Grants DFG-RO 3671/6-2 to P.R. and (SFB 834 to PMB and IF).

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Author notes

  1. Peter M. Benz and Peter Rosenberger have contributed equally.

Authors and Affiliations

  1. Department of Anesthesiology and Intensive Care Medicine, Universitätsklinikum Tübingen, Hoppe-Seyler-Straße 3, 72076, Tübingen, Germany

    David Köhler, Lothar P. Klammer, Judith M. Roth, Tiago F. Granja, Andreas Straub & Peter Rosenberger

  2. Division of Clinical Chemistry, Department of Internal Medicine, University Hospital, Tübingen, Germany

    Rainer Lehmann

  3. Institute for Vascular Signalling, Centre for Molecular Medicine, Johann Wolfgang Goethe University, Frankfurt, Germany

    Sofia-Iris Bibli, Ingrid Fleming & Peter M. Benz

  4. German Center of Cardiovascular Research (DZHK), Partner Site Rhein-Main, Frankfurt am Main, Germany

    Sofia-Iris Bibli, Ingrid Fleming & Peter M. Benz

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  1. David Köhler
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Contributions

DK: designed research, performed experiments, analyzed data, and wrote the manuscript. S-IB: performed experiments and analyzed data. Lothar Klammer: performed experiments. JMR: performed experiments. RL: analyzed data. TG: performed experiments and analyzed data. IF: designed research and analyzed data. AS: elaborate experiments, analyzed data, and wrote the manuscript. PMB: performed experiments, analyzed data, and wrote parts of the manuscript. PR: designed research and wrote the manuscript.

Corresponding author

Correspondence to Peter Rosenberger.

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Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical standards

All animal studies have been approved by the appropriate ethics committee and have, therefore, been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments (Institutional Animal Care and Use Committee of the Tübingen University Hospital and the Regierungspräsidium Tübingen). After written informed consent was obtained from each person studied, blood was withdrawn and bloodcells isolated from whole blood. The withdrawl was approved by the Institutional Review Board of Tübingen University (ethics committee). Therefore, all studies were human blood was used were in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

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Supplemental Fig. 1 Flow cytometric detection of the leukocyte population (upper dot plot) and platelets as well as platelet–platelet/platelet–leukocyte aggregates (lower dot plot) a)

Upper dot plot: Granulocytes, monocytes, and lymphocytes are detected in specific regions. The lower dot plot indicates platelets as well as platelet/platelet–granulocyte aggregates b and c) Dilution series determines maximal possible VASP phosphorylation at site serine 153 and serine 235 using the well-known potent phosphorylating agent PGE1 (n=4 per group) in murine thrombocytes and neutrophil granulocytes. d and e) Maximal possible VASP phosphorylation at site serine 153 and serine 235 using PGE1 (n=4 per group) in murine platelets and neutrophil granulocytes based on %-gated (TIFF 729 kb)

Supplemental Fig. 2 Negative controls for myocardial tissue stainings.

(TIFF 2076 kb)

Supplemental Fig. 3 Verification of successful generation of VASP-chimeric animals a)

Western Blot analysis of chimeric mice following bone marrow transplantation demonstrating VASP expression in the myocardial tissue and blood of WT → WT transplanted animals, myeloid WT into VASP-/- animals (WT → VASP-/-), and VASP-/- in WT animals (VASP-/- → WT), and VASP-/- → VASP-/-transplanted control animals (pooled samples of n=6/ group). (TIFF 447 kb)

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Köhler, D., Bibli, SI., Klammer, L.P. et al. Phosphorylation of vasodilator-stimulated phosphoprotein contributes to myocardial ischemic preconditioning. Basic Res Cardiol 113, 11 (2018). https://doi.org/10.1007/s00395-018-0667-0

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  • DOI: https://doi.org/10.1007/s00395-018-0667-0

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