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Evidence for an intracellular localization of the adenosine A2B receptor in rat cardiomyocytes

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Abstract

Protection achieved by ischemic preconditioning is dependent on A2B adenosine receptors (A2BAR) in rabbit and mouse hearts and, predictably, an A2BAR agonist protects them. But it is controversial whether cardiomyocytes themselves actually express A2BAR. The present study tested whether A2BAR could be demonstrated on rat cardiomyocytes. Isolated rat hearts experienced 30 min of ischemia and 120 min of reperfusion. The highly selective, cell-permeant A2BAR agonist BAY60-6583 (500 nM) infused at reperfusion reduced infarct size from 40.4 ± 2.0% of the risk zone in control hearts to 19.9 ± 2.8% indicating that A2BAR are protective in rat heart as well. Furthermore, BAY60-6583 reduced calcium-induced mitochondrial permeability transition in isolated rat cardiomyocytes. A2BAR protein could be demonstrated in isolated cardiomyocytes by western blotting. In addition, message for A2BAR was found in individual cardiomyocytes using quantitative RT-PCR. Surprisingly, immunofluorescence microscopy did not show A2BAR on the cardiomyocyte’s sarcolemma but rather at intracellular sites. Co-staining with MitoTracker Red in isolated cardiomyocytes revealed A2BAR are localized to mitochondria. Western blot analysis of a mitochondrial fraction from either rat heart biopsies or isolated cardiomyocytes revealed a strong A2BAR band. Thus, the present study demonstrates that activation of A2BAR is strongly cardioprotective in rat heart and suppresses transition pores in isolated cardiomyocytes, and A2BAR are expressed in individual cardiomyocytes. However, surprisingly, A2BAR are present in or near mitochondria rather than on the sarcolemma as are other adenosine receptors. Because A2BAR signaling is thought to result in inhibition of mitochondrial transition pores, this convenient location may be important.

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Acknowledgments

We thank Thomas Krahn and Barbara Albrecht (Bayer HealthCare Wuppertal, Germany) for providing BAY60-6583 and performing the concentration measurements (see supplemental material). We thank Dr J. Linden, University of Virginia, Charlottesville, VA, for the HEK293-A2BAR cells, and Dr H. Eltzschig, University of Colorado, Denver, CO, for the A2BAR knockout mice (see supplemental material). The study was in part supported by grants from the National Institutes of Health Heart, Lung and Blood Institute HL-20648 (T.K., J.M.D.) and the Deutsche Forschungsgemeinschaft (T.K.).

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Authors and Affiliations

  1. Department of Cardiology, Ernst-Moritz-Arndt University, Greifswald, Germany

    Karina Grube, Julia Rüdebusch, Thomas Böckenholt, Katrin Zimmermann & Stephan B. Felix

  2. Department of Anatomy, Ernst-Moritz-Arndt University, Greifswald, Germany

    Tobias Müller

  3. Department of Anesthesiology, University of North Carolina, Chapel Hill, NC, USA

    Zhelong Xu

  4. Cardiovascular Division, King’s College London, London, UK

    Friederike Cuello

  5. Department of Physiology, University of South Alabama, Mobile, AL, USA

    Xiulan Yang, Michael V. Cohen & James M. Downey

  6. Department of Medicine, University of South Alabama, Mobile, AL, USA

    Michael V. Cohen

  7. Clinical Pharmacology Unit, Addenbrooke’s Hospital, University of Cambridge, Box 110, Cambridge, CB2 2QQ, UK

    Carmen Methner & Thomas Krieg

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  1. Karina Grube
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  2. Julia Rüdebusch
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Correspondence to Thomas Krieg.

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Grube, K., Rüdebusch, J., Xu, Z. et al. Evidence for an intracellular localization of the adenosine A2B receptor in rat cardiomyocytes. Basic Res Cardiol 106, 385–396 (2011). https://doi.org/10.1007/s00395-011-0151-6

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