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NECA at reperfusion limits infarction and inhibits formation of the mitochondrial permeability transition pore by activating p70S6 kinase

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Abstract

The A1/A2 adenosine agonist 5′-(N-ethylcarboxamido) adenosine (NECA) limits infarction when administered at reperfusion. The present study investigated whether p70S6 kinase is involved in this anti-infarct effect. Adult rat ventricular myocytes were isolated and incubated in tetramethylrhodamine ethyl ester (TMRE, 100 nM), which causes cells to fluoresce in proportion to their mitochondrial membrane potential. A reduction in TMRE fluorescence serves as an indicator of collapse of the mitochondrial transmembrane potential. Cells were subjected to H2O2 (200 µM), which like ischemia induces loss of mitochondrial membrane potential. Fluorescence was measured every 3 min and to facilitate quantification membrane potential was arbitrarily considered as collapsed when fluorescence reached less than 60% of the starting value. Adding NECA (1 mM) to the cells prolonged the time to fluorescence loss (48.0 ± 3.2 min in the NECA group versus 29.5 ± 2.2 min in untreated cells, P < 0.001) and the mTOR/p70S6 kinase inhibitor rapamycin (5 nM) abolished this protection (31.3 ± 3.4 min). Since cyclosporine A offered similar protection, mitochondrial permeability transition pore formation is a likely cause of the H2O2-induced loss of potential. The direct GSK-3β inhibitor SB216763 (3 µM) also prolonged the time to fluorescence loss (49.2 ± 2.1 min, P < 0.001 versus control), and its protection could not be blocked by rapamycin (42.2 ± 2.3 min, P < 0.001 versus control). NECA treatment (100 nM) of intact isolated rabbit hearts at reperfusion after 30 min of regional ischemia decreased infarct size from 33.0 ± 3.8% of the risk zone in control hearts to 11.8 ± 2.0% (P < 0.001), and rapamycin blocked this NECA-induced protection (38.3 ± 3.7%). A comparable protective effect was seen for SB216763 (1 µM) with infarct size reduction to 13.5 ± 2.3% (P < 0.001). NECA treatment (200 nM) of intact rabbit hearts at reperfusion also resulted in phosphorylation of p70S6 kinase more than that seen in untreated hearts. This NECA-induced phosphorylation was blocked by rapamycin. These experiments reveal a critical role for p70S6 kinase in the signaling pathway of NECA’s cardioprotection at reperfusion.

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Acknowledgments

We thank Karin Lissek for technical assistance and Sandra Grugel for myocyte preparation. This study was supported in part by grants from the Department für kardiovaskuläre Medizin (T.K.) and the Forschungsverbund Molekulare Medizin (T.K.) of the University Greifswald, and HL-20648 (J.M.D.) and HL-50688 (M.V.C.) from the National Heart, Lung, and Blood Institute of NIH. K.F. was supported by a grant from BMBF/NBL-3.

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Authors and Affiliations

  1. Department of Cardiology, Ernst-Moritz-Arndt University Greifswald, Loefflerstr. 23, 17487, Greifswald, Germany

    Karina Förster, Ina Paul, Alexander Staudt, Stephan B. Felix & Thomas Krieg

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

    Natalia Solenkova, Michael V. Cohen & James M. Downey

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

    Michael V. Cohen

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  1. Karina Förster
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  2. Ina Paul
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  3. Natalia Solenkova
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  4. Alexander Staudt
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  5. Michael V. Cohen
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  6. James M. Downey
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  7. Stephan B. Felix
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  8. Thomas Krieg
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Correspondence to Thomas Krieg.

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Returned for 1st revision: 3 November 2005 1st revision received: 3 February 2006

Returned for 2nd revision: 23 February 2006 2nd revision received: 1 March 2006

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Förster, K., Paul, I., Solenkova, N. et al. NECA at reperfusion limits infarction and inhibits formation of the mitochondrial permeability transition pore by activating p70S6 kinase. Basic Res Cardiol 101, 319–326 (2006). https://doi.org/10.1007/s00395-006-0593-4

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  • DOI: https://doi.org/10.1007/s00395-006-0593-4

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